Process Biochemistry 44 (2009) 481485

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Process Biochemistry
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Size exclusion chromatography of zein and xanthophylls Optimization of operating parameters

Aniket Kale *, Munir Cheryan
University of Illinois at Urbana Champaign, Agricultural Bioprocess Laboratory, 1302 W. Pennsylvania Avenue, Urbana, IL 61801, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 March 2007 Received in revised form 16 December 2008 Accepted 17 December 2008 Keywords: Process chromatography Optimization SEC Corn Zein Xanthophylls

Ethanol-soluble components of corn (zein and xanthophylls) were separated and puried by size exclusion chromatography. Aqueous ethanol was used as the solvent for the entire process from extraction through chromatography. The effect of operating and design parameters, such as temperature, ow rate, loading mass, loading volume, column height and diameter, on productivity and resolution of the components was studied. Using a one-variable-at-a-time (OVAT) approach with 1 cm 60 cm columns, optimum conditions were determined to be 40 8C temperature, 0.25 mL/min ow rate, volume loading of 22 mL corn extract, mass loading at 70 g/L of corn extract. All components could be resolved with base line separation with a 240 cm column length. Column diameter did not affect separation, implying linear scalability with constant ow distribution. Published by Elsevier Ltd.

1. Introduction Industrial chromatography is well established for producing puried products in the pharmaceutical and biotechnology industries. It is nding increasing use in the food industry for specialized applications such as production of nutraceuticals [1], demineralization of sugars [2], amino acids purication [3], production of pectin and its derivatives [4], decolorization [5] organic acids purication [6] and whey protein purication [7]. Enrichment of fructose for the production of high-fructose corn syrups is probably the largest single application [8]. Most of these applications use either ion-exchange or adsorption chromatographic methods. However, these applications are based on aqueous systems. Further, these applications have different approaches and unit operations for large molecules (such as proteins) and small molecules (such as carotenoids, organic acids). This paper describes the separation of a complex mixture of proteins, color pigments and impurities (a complex mixture of non-protein, non-carotenoid compounds) in an ethanol extract of corn by size exclusion chromatography. The proteins are a group of prolamines known collectively as zein with a molecular weight of 1048 kDa. Zein has a wide variety of applications from biodegradable plastics to wound dressings and shellac replacer [9]. The pigments are yellow-orange colored oxygenated carote-

* Corresponding author. Tel.: +1 217 898 3895; fax: +1 217 333 9332. E-mail address: akale@uiuc.edu (A. Kale). 1359-5113/$ see front matter . Published by Elsevier Ltd. doi:10.1016/j.procbio.2008.12.015

noids known as xanthophylls, primarily lutein and zeaxanthin, with a molecular weight of 565 Da. These xanthophylls have specic uses in mitigating eye diseases such as age-related macular degeneration [10]. The impurities are a group of compounds of unknown composition with a molecular weight of less than 10 kDa as determined by MALDI-MS. The challenge was that the extraction of these compounds from corn, and the subsequent separation and purication, is done in 70% (v/v) aqueous ethanol. Further, neither zein nor the carotenoids are soluble in water and zein has unique solubility characteristics [9] which complicate the separation. Size exclusion chromatography for industrial separations is used primarily for fractionation and desalting [11]. In the fractionation mode, the feed solids go through the pores and elute in an order based on their size, with the largest compound eluting rst. The pore size of the stationary phase/resin needs to be at least four times the size of the compound and, in addition, for good resolution, the ratio of the molecular weights of the fractionated solutes should be at least 2 [12]. In the desalting mode, the required compound is bigger than the pore and elutes in the void volume while the smaller molecule elutes later. This method has been used to desalt proteins after ion exchange and for removing ethanol from blood plasma [13]. In our case, we selected a stationary phase containing pores of the appropriate molecular weight cut off ($5 kDa). The proteins were excluded in the void volume and were puried in the desalting mode, while the impurities and xanthophylls were separated over the length of the column in the fractionation mode [14]. Thus, three pure products

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were obtained in a single step: zein, impurities and xanthophylls. This method of separation maintains the proteins in their native state which could be important in high-value applications of zein. It also minimizes degradation of xanthophylls by avoiding exposure to heat, light, oxygen and UV light. Thus, our work is unique because rstly it is a prep-scale application of aqueous ethanol size exclusion chromatography and secondly because it uses both the fractionation and desalting modes of size exclusion chromatography to purify two valuable compounds from a complex mixture in a single step. The focus of this study was to identify and optimize important design and operating parameters. Design parameters such as column dimensions determine process economics and control selectivity and resolution. The effects of operating parameters such as temperature, ow rate, volume loading and mass loading on throughput and resolution were studied and optimized. The acceptable purity and yield for the existing markets were about 90%.
2. Materials and methods The reagent, equipment details for these experiments and the corn extract preparation protocol and the analytical procedures have been described in detail in previous publications [14]. 2.1. Experimental variables The eluent in all experiments was aqueous ethanol. Column temperature was maintained by circulating water at the appropriate temperature through the column jacket. The effect of column height was studied by connecting several columns in series. Volume loading experiments were performed using various commercial sample loops obtained from Alltech Associates (Deereld, IL). Two or more loops were connected in those cases where a single loop was not available for a particular sample volume. For large volumes, loops were made from the tubing. Mass loading experiments were performed using dilutions of a pre-concentrated extract. The ethanol extract was concentrated using a reverse osmosis membrane (SW30, Dow/FilmTec, Edina, MN) as described by Tsui and Cheryan [15] in an Amicon dead-end stirred cell at 50 8C and 400 psi to a total solids of 220 g/L. This concentrate was diluted with 70% ethanol as needed and injected into the column. 2.2. Performance parameters The resolution of zein (peak 1: void volume peak in the 280 nm chromatogram) and xanthophylls (peak 2: exclusion volume peak in the 450 nm chromatogram) were determined by using Eq. (1):  RS 2 V R2 V R1 W H1 W H2  (1)

Fig. 1. Effect of temperature on the separation of corn extract on a 60 cm LH-20TM column. Eluent was 70% (v/v) ethanol at 1 mL/min with a TricornTM 1 cm 60 cm column.

the LH-20TM resin [16]. All other variables were kept constant, i.e., 1 mL/min ow rate of 70% ethanol and 1 mL injection. Fig. 1 shows the effect of temperature on resolution. There was an increase in RS and productivity with temperature, conceivably due to a reduction in peak tailing and mobile phase viscosity. An increase in temperature narrowed the peak and reduced the peak tail and thus the elution time by approximately 15% [14]. This observation is consistent for entropy-driven separations such as size exclusion chromatography [17]. Solute distribution is controlled by the entropy change and enthalpy change is negligible. Thus, temperature does not have a direct inuence on the separation, although it affects size exclusion separations indirectly through its inuence on the viscosities of the mobile phase and the feed, which in turn could affect mass transfer rate [18]. Hence, the highest temperature that the stationary phase can withstand would be the best temperature. However, in practice, the cost of maintaining the column at that temperature and possibly reducing the life of the stationary phase has to be taken into consideration before selecting the temperature. 3.1.2. Flow rate The maximum ow rate is limited by column pressure drop considerations. The maximum pressure drop was calculated by the BlakeKozenyCarman equation for packed beds. Based on the manufacturers recommendation for the maximum linear ow rate of 0.2 cm/s for the LH-20TM resin for a 14 cm column [16], the maximum pressure drop across the column was 5.2 bar (75 psi). In this calculation, the porosity of the column was 0.25 as observed from the void volume and elution of zein, the average particle size was 70 mm from the manufacturers specications and viscosity of the mobile phase (70% ethanol) was 1.4 cP at room temperature. In general, the operating ow rate is 7090% of the maximum allowed ow rate to account for changes in viscosities of the feed and mobile phase or other factors. We have used up to 70% of the maximum for our experiments. Fig. 2 shows that RS decreases as ow rate increases, due probably to broadening of the xanthophylls peak. Improving mass transfer is the key to enhancing the resolution, and mass transfer is affected by particle size and ow distributions. Productivity, on the other hand, increases almost linearly with ow rate. The effect of ow rate through the column on separation efciency is best illustrated by the Van Deemter equation. The optimum ow rate occurs at the minimum plate height. However, this ow rate is generally too low and practically impossible to be used on a process scale. Thus in practice, the ow rate would be greater than the optimum implying sub-optimal resolution.

where VR1 is the retention volume of peak 1, VR2 is the retention volume of peak 2, WH1 is the width of peak 1 and WH2 is the width of peak 2. Yield of zein is dened as the weight of zein in the zein fraction to the weight of zein loaded on the column. Purity of zein is dened as the weight percent of zein in the zein fraction. Productivity of the process is dened as the amount of pure zein produced per unit resin volume per unit time (g/L per day).

3. Results and discussions The basis or the anchor data point for the optimization experiments was a sample injection of 1 mL of corn extract containing 7 g/L of zein and 14 g/L of total solids at a ow rate of 1 mL/min of 70% aqueous ethanol at room temperature. The data for the fractions collected in such an experiment has been explained in detail in prior work [14]. The effect of operating variables (temperature, ow rate) design variables (column diameter and length) and column loading (loading volume and mass) were studied using the one-variable-at-a-time (OVAT) approach and are discussed as follows. 3.1. Operating variables 3.1.1. Temperature Experiments were conducted with the 1 cm diameter column at 5 8C intervals between 25 8C and 40 8C which is the upper limit of

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Fig. 2. Effect of ow rate on the separation of corn extract on a 60 cm LH-20TM column. Eluent was 70% ethanol and 25 8C with a TricornTM 1 cm 60 cm column.

Fig. 3. Effect of column length on resolution and productivity of corn extract components. Eluent was 1 mL/min of 70% ethanol at 40 8C.

4. Design variables 4.1. Column diameter In this study, columns of different internal diameters (1 cm, 1.5 cm, and 2.5 cm) were used under similar operating conditions (room temperature, eluent ow rate of 1 mL/min of 70% ethanol). The volumetric ow rate and loading volume were scaled up in proportion to the column volume. The loading of 5 mL on a 1 cm column at 1 mL/min was translated to an 18.75 mL injection at 2.25 mL/min for the 1.5 cm diameter column and 50 mL injection at 6.25 mL/min for the 2.5 cm diameter column. The ow rates were calculated based on the column diameter keeping the linear ow constant. The injection volumes were 10% of the total column volume. The resolution and productivity was relatively unaffected by column diameter implying a linear scalability of the separation process. 4.2. Column height In this study, several columns of 1 cm i.d. were connected in series under equivalent standard operating conditions (room temperature, eluent ow rate of 1 mL/min of 70% ethanol, 5 mL corn extract injection). Zein elutes at the void volume of the column, which was about 25% of the total column volume, while the xanthophylls elute as a group after 85% of the column volume with all column heights studied [14]. Above 240 cm, the impurities peak resolved into its components and also resulted in purer xanthophylls peak. In general, elution with 130% of the column volume completely eluted all the components in the column irrespective of column height. Fig. 3 shows the effect of column height on resolution and productivity. Increasing the column height above 120 cm resulted in baseline separation of zein and impurities (RS > 1.5). A column length of 240 cm would result in baseline separation of impurities and xanthophylls. Increasing the length of a SEC column increases total internal pore volume, which increases the time available for attaining equilibrium. In our specic case, this would retard the impurities fraction resulting in a purer zein fraction, while simultaneously retarding the xanthophylls fraction and resulting in a xanthophylls fraction with fewer impurities. However, it is also possible that longer columns would lead to crushing of the beads in the lower part of the column. Productivity is inversely proportional to the column height, while the resolution of the xanthophylls and impurities is directly proportional to the column height.

4.3. Column loading 4.3.1. Loading volume This work is a novel attempt to utilize size exclusion resin with functional groups for adsorption to work in partition and fractionation modes. The rules of thumb for less than 5% loading for maximum resolution are not the optimum in our case. Due to the effect of aqueous ethanol mobile phase versus pure water, the void volume properties have changed. Any loading beyond the void volume will result in overloading the zein. On the other hand, the resin used has lipophilic hydrophilic moieties on the surface causing a weak binding of the xanthophylls and elution over one column volume and even higher for higher loading. Fig. 4 shows the chromatograms obtained at various injection volumes, keeping all other conditions constant. The maxima of the zein and the impurity peaks correspond to elution volumes of 15 mL and 35 mL, respectively. The impurity peaks are relatively sharper at higher loading indicating that a concentrated product was eluting. A similar effect was observed by Katti and Guichon [19] and was attributed to the displacement effect leading to concentration and compression of the component. The impurity peak front becomes more prominent at loading volumes greater than 5 mL. Fractions Fr1 through Fr5 from Fig. 4 were collected and analyzed for yield and purity of zein. The effect of loading volume on the yield and purity of Fr1 is shown in the inset of Fig. 4. An increase in loading volume A caused a slight increase in the purity and a large reduction in yield. The optimum loading volume for the 60 cm column was 5 mL at which the yield and purity were 91% and 88%, respectively. The effect of loading volume on the separation of impurities and xanthophylls is shown in Fig. 4. The xanthophylls peak shifts to higher elution volumes with an increase in loading volume. However, the xanthophylls peak front is almost constant for all loading volumes. The proportion of xanthophylls that are totally devoid of impurities increases with loading volume. Since the peak elution volume for the xanthophylls peak is shifting towards the right and that of the impurities is almost constant at 35 mL (Fig. 4), the resolution between impurities and xanthophylls increases with an increase in loading volume. 4.3.2. Mass loading In this series of experiments, sample loading volume was maintained at 5 mL based on the volume loading studies. Five fractions were collected in each run and the total solids and protein were determined for each fraction. Fig. 5 shows the concentration proles of zein, impurities and xanthophylls at different mass

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Fig. 4. Effect of the column loading volume on the elution proles of zein, impurities and xanthophylls. Eluent was 70% ethanol at 1 mL/min and 25 8C with a TricornTM 1 cm 60 cm column. Inset: effect of loading volume on the yield and purity of Fraction 1 (closed points: yield; open points: purity).

Fig. 5. Effect of loading concentration on the separation of zein and impurities. Eluent was 70% ethanol at 1 mL/min and 25 8C with a TricornTM 1 cm 60 cm column. Inset: effect of loading mass of the feed on yield and purity of zein in Fraction 1 (open points: purity; closed points: yield).

loadings. The rst zein peak broadens at higher mass loading, similar to the volume loading study. This may be caused by higher feed viscosity which reduces mass transfer rates and leads to dispersion of zein into the eluting impurities. The yield and purity of Fraction 1 are shown in the inset of Fig. 5. Purity increases linearly with loading concentration. The yield increases up to a mass loading of 70 g/L and then decreases. Thus, the best conditions are injection of a 5 mL sample containing 70 g/L of solids. This will result in 85% yield and 95% purity of zein. The concentration of the compounds in the feed has a major impact on the economics of chromatographic separations. Higher concentrations reduce solvent usage which in turn reduces the size of equipment and the cost of operation. The rule of thumb for the maximum loading concentration of globular proteins on an SEC column is about 70 g/L [20]. Fig. 5 also shows the chromatograms of the impurities and xanthophylls as a function of mass loading. The maxima of the zein,

impurities and xanthophylls peaks are at elution volumes of 15 mL, 35 mL and 45 mL, respectively. The impurities and xanthophylls peaks are non-skewed and normal-shaped towards the statistical mode with long at fronts and tails. The peak overlap is symmetrical for all loading concentrations. It appears that loading concentration had little or no effect on separation of impurities and xanthophylls. 5. Conclusions Chromatographic unit operations are generally operated in a sequence of several steps such as capture and isolation, intermediate purication and polishing. Each results in a gain in purity but a loss in yield [8]. In most cases, it is difcult to obtain both high yields and high purity simultaneously. However, we have obtained both high purity of the protein (>90%) and high yields (>90%). In addition, we have also simultaneously obtained

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an additional high-value coproduct in a relatively pure form (xanthophylls). There is no denaturation of the proteins and no need for buffers and salts in the process, thus further reducing costs. The solvent used in this process is aqueous ethanol, which is available in-house in dry-grind ethanol plants which also has abundant quantities of the raw material (corn). In large-scale operations, if pure xanthophylls devoid of impurities are the desired product, longer columns should be used since column height enhanced the overall resolution of impuritiesxanthophylls and provided higher loading capacities for zein purication. The absence of any effect on resolution with an increase in column diameter indicated that the system is linearly scalable. The highest temperature compatible with the stationary phase should be used. The maximum ow rate is restricted by the loss in resolution and high pressure drops. From the volume and mass loading study, an increase in the volume loading shifted the xanthophylls peaks to the right further improving the xanthophyllsimpurities separation but this also means an increase in the cycle time. Mass loading did not affect the separation of impurities and xanthophylls. The maximum volume of corn extract that could be loaded on to a 1 cm 60 cm column was about 5 mL of corn extract concentrate containing up to 70 g/L total solids (35 g/L zein). However, in terms of manufacturing process economics, this separation strategy may not be the best option. Optimum productivity of zein at 4 g/L per day is low by manufacturing standards. With an optimistic estimate of 500 uses of the resin and its conservative cost of USD 500/L, the productivity transforms into 4 g/USD for resin implying 250 USD/kg zein which is perhaps high. Hence, a detailed techno-economic analysis was presented by the authors in prior work [14]. Acknowledgements The project was supported by the Illinois Corn Marketing Board, the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service; grant number 200435503-14116 and the University of Illinois Research Board with an equipment grant. Financial support in the form of

scholarships was provided to AK by AACC International, St. Paul, MN, and the Department of Food Science & Human Nutrition, University of Illinois at Urbana-Champaign. References
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