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The toroidal column using a zigzag pattern has been improved in both retention of the stationary phase and peak resolution. To further improve the retention of stationary phase and peak resolution, a series of novel geometric designs of tubing (plain, mid-clamping, flattened and flat-twisted tubing) was evaluated their performance in CCC. The results showed that the tubing which was flattened vertically against centrifugal force (vert-flattened tubing) produced the best peak resolution among them. Using vert-flattened tubing a series of experiments was performed to study the effects of column capacity and sample size. The results indicated that a 0.25 ml capacity column is ideal for analysis of small amount samples.
Column design is the heart of the chromatographic system that leads to the successful separation. Therefore, it is surprising to find that until now only few publications have been dedicated to the column design for countercurrent chromatography, a support-free chromatographic technique, which has been widely used for the separation and purification of natural products [1, 2].
Geometries of tubing and its mounting patterns lead to different chromatographic behaviors and separation efficiency. In the previous CCC studies, there are only six papers involved in the modified tubing shape and its mounting patterns. Degenhardt et al. compared of performance of custom-made rectangular tubing with or without twisting and found that the twisted rectangular tubing yielded 15-30 % higher efficiency in separation of natural products than that of the conventional standard multilayer coil . Cross-pressed tubing has been introduced to improve the separation with the spiral tube assembly for HSCCC , where resolution (Rs) has been remarkably increased compared with traditional plain tubing. Recently, flat-twisted tubing mounted in the spiral tubing support in HSCCC produced efficient separation of protein samples .
In our previous study, the toroidal column was designed to triangular helical pattern and zigzag pattern to improve separation efficiency for the hydrostatic centrifugal CCC system. These tubing designs improved peak resolution (Rs) mainly due to higher retention level of the stationary phase compared with traditional toroidal coiled column [6, 7]. Rs of the zigzag toroidal column was further improved by modifying the tubing geometry.
Recently we have developed a novel toroidal zigzag column (fig. 1), which remarkably increased the retention of stationary phase, and by clumping a middle portion of each loop (mid-clamping tubing) the separation was further improved. In present study, a series of experiments were performed using a set of modified zigzag toroidal tubing to compare their performance in terms of retention of the stationary phase and peak resolution at various flow rates (fig. 2).
The present study uses a rotary-seal-free centrifuge fabricated by Pharma-Tech Research Corporation, Baltimore, Maryland, USA. It is equipped with an aluminum rotary plate measuring about 34 cm in diameter to hold a toroidal coil separation column on its periphery. The column is made by hooking a set of modified tubing (fluorinated ethylene propylene tubing from Zeus Industrial Products, Orangeburg, SC, USA) onto the screws upstanding over the rotary plate forming the same zigzag pattern (fig. 2). These tubing modifications include tubing clamped at the middle portion of the loop either parallel or perpendicular to centrifugal force (para-mid-clamping tube or vert-mid-clamped tubing, respectively), flattened tubing parallel or perpendicular to centrifugal force (para-flattened tube or vert-flattened tubing, respectively) and twisted flat tubing (flat-twisted tubing). Each terminal of the toroidal column is connected to flow tubes with a set of tubing connectors (Upchurch Scientific, Palm Spring, CA, USA) as shown in Fig. 1. These flow tubes are put together and passed through the center of the central shaft downward and the hollow horizontal shaft of a miter gear, then led upward into the vertical hollow tube support, and finally exit the centrifuge from the center of the upper plate where they are tightly held with a pair of clamps.
1-Butanol of HPLC grade was purchased from Fisher Scientific, Fair Lawn, NJ, USA and acetic acid of analytical grade from Mallinckrodt Chemicals, Phillipsburg, NJ, USA. Dipeptide samples including tryptophyl-tyrosine (trp-tyr) and valyl-tyrosine (val-tyr) were obtained from Sigma Chemicals, St. Louis, MO, USA.
In the present study, a typical two-phase solvent systems, 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), was used to separate the dipeptide samples. Solvent mixture was thoroughly equilibrated in a separatory funnel by repeating vigorous shaking and degassing several times, and the two phases separated shortly before use. The sample solution was prepared by dissolving 25 mg of trp-tyr (Kup/lp = 1.69) and 100 mg of val-tyr (Kup/lp = 0.53) in 20 ml of the upper phase of BAW, and 50 μl was charged in each run .
In each separation, the separation column was entirely filled with the stationary phase, either upper or lower phase, followed by sample injection, and the column was rotated at 1000 rpm while the mobile phase was pumped into the coiled column at a given flow rate. The effluent from the outlet of the coiled column was continuously monitored with a Uvicord IIS (LKB, Stockholm, Sweden) at 280 nm and the elution curve was traced using a stripped-chart recorder (Pharmacia, Stockholm, Sweden). After the desired peaks were eluted, the run was stopped and the column contents were collected into a graduated cylinder by pressured air to determine the volume of the stationary phase retained in the column. The retention of the stationary phase was computed by dividing the volume of the retained stationary phase with the total column volume.
The partition efficiency of separation column in each run was evaluated by computing theoretical plate number (N) for each peak and peak resolution (Rs) between the peaks using the following conventional equations:
In order to make a fair comparison between the present results and the previous data obtained from the standard column (capacity V2) with a larger capacity, the peak resolution (Rs) obtained from the present flat-twisted spiral tube assembly (capacity V1) was adjusted using the following equation:
where Rs-a1 is the adjusted peak resolution.
And the peak resolution was similarly compared at the same column length as follows:
where Rs-a2 is the adjusted peak resolution and L indicates the column length.
In the past plain tubing was mainly used as the separation coil for HSCCC. In this tubing geometry, the centrifugal force acting perpendicularly to the axis of tubing gives the identical effect in all directions. However, if the shape of tubing was modified into a flat form, the tubing has maximum and minimum internal diameters and the centrifugal force produces different effects on hydrodynamic motion of the two phases according to its acting direction, parallel (across the minimum diameter forming minimum interface area) and perpendicular (across the maximum diameter forming maximum interface area) to the flat surface. The mutual relationship between the cross- sectional area and minimal and maximum internal diameters of flat tubing was studied by squashing a 5 mm I.D. O-ring between two parallel straightedges. The results of this simulating model study are shown in Fig. 3 where both minimum and maximum internal diameters, internal area and their ratio are plotted. As the O-ring is squashed, the area of the internal area is decreased and the ratio between the maximum internal diameter and the area (M/A) is sharply increased. In the actual separation with two phase solvent system in the flat tubing, M represents the interfacial area when the centrifugal force is acting perpendicular to the surface of the flat tubing and the A, the volume of the two phases forming the interface. Since the partition efficiency increases with the interface area per volume of the two phases in the tubing, the best result is expected by applying the centrifugal force perpendicularly to the flat surface of the tubing provided that the retention of the stationary phase is adequately maintained under a low flow rate.
A series of experiments was performed on separation of dipeptides with the polar BAW solvent system in centrifugal countercurrent chromatography. Table 1 summarizes the retention of stationary phase, peak resolution and N in 6 sets of zigzag toroidal tubing each with different geometry and orientation including plain tubing, tubing clamped at the middle of each loop in two different directions, flat tubing mounted in different directions and flat-twisted tubing. In both clamped and flattened tubing, vert- indicate the centrifugal force acting perpendicular to the flatted surface, and para- indicates that acting parallel to the flattened surface. When the plain tubing is modified to mid-clamped or flattened tubing, the capacity decreases while the length increases appreciably.
In all groups the retention of stationary phase increases as the flow rates is decreased rate (Table 1). In the plain tubing, the retention of stationary phase with lower phase mobile was higher than that with upper phase mobile. And when mid-clamping tubing was tested, the retention of stationary phase with both lower and upper mobile phases was very similar. However, in the flattened tubing the retention of stationary phase with upper phase mobile was better than that with lower phase mobile and, as expected, the retention of stationary phase remarkably decreased. The stationary phase retention of para-mid-clamping and para-flattened tubing was better than that of vert-mid-clamping and vert-flattened tubing, respectively. When lower phase was mobile phase, retention of stationary phase using plain tubing is the best at a given flow rate. When upper phase was mobile phase, however, both plain and para-flattened tubing designs can produce good retention of the stationary phase even at the high flow rate of 0.05 ml/min while the stationary phase retention of vert-flattened tubing was only 12.03 % at the same flow rate. The retention of stationary phase with the flat-twisted tubing was slightly better than that with the vert-flattened tubing.
The performance of each group of tubing in Table 1 is compared in terms of peak resolution (Rs) and theoretical plate number (N) by adjusting the data at the same capacity (Rs-a1) and the same length (Rs-a2) of tubing, respectively, according to Eqs. 3 and 4 described earlier. All groups was tested at several flow rates ranging from 0.005 to 0.05 ml/min. Fig. 4 illustrates the variations of adjusted resolution at the same column capacity at the flow rates from 0.005 to 0.05 ml/min in the dipeptide separation using 5 different types of the zigzag toroidal tubing. All the results were adjusted based on the Eq. 3. With increased flow rate, the adjusted resolution (Rs-a1) sharply decreased. When the lower phase is mobile phase, the Rs-a1 using flat-twisted was the best at 2.53 and the para-mid-clamping tubing was the worst at the high flow rate of 0.05 ml/min. When the flow rate for para-mid-clamping tubing was decreased to 0.005 ml/min, the Rs-a1 was sharply increased reaching 5.13, the highest level among all groups, apparently due to the improved retention of stationary phase to 39.23 % and its broad interface. When upper phase was mobile, the Rs-a1 was lower than that of lower phase mobile due to the low retention of stationary phase and at a flow rate of 0.05 ml/min, the retention dropped down to 12.03%, showing single peak in the chromatogram. But when the flow rate was decreased to 0.005 ml/min, the retention of stationary phase was sharply increased to 43.5% with a high peak resolution at 3.50 (Table 1). Flat-twisted, vert-flattened and vert-mid-clamping tubing all show excellent separation (Table 1).
When the data were adjusted and compared at the same tubing length, the results was very different from the results adjusted at the tubing capacities. All the results were adjusted based on the Eq. (4). Though adjusted resolution (Rs-a2 in Table 1) still increased with decreased flow rate, the Rs-a2 using vert-flattened tubing was not the best at the lower flow rate. When the lower phase is mobile phase, the para-flattened tubing had a good performance of RS-a2 = 2.23 at 0.05 ml/min of flow rate. But when flow rate was decreased to 0.005 ml/min, the Rs-a2 of vert-mid-clamping was the best. Vert-mid-clamping tubing showed also very good performance, and Rs-a2 was 1.71 at higher flow rate and 3.58 at lower flow rate.
Since two different results were obtained in the above comparative method, a series of experiments were designed to compare the Rs at the similar peak retention time between the plain, vert-flattened and flat-twisted tubing (Fig. 5). The peak retention time was adjusted based on the column capacity by applying the different flow rates according to the column capacity. Fig. 5A to Fig. 5C show the results of plain tubing, vert-flattened tubing and flat-twisted tubing, respectively. The result revealed that the vert-flattened tubing at a lower flow rate yields slightly better peak resolution than those of plain and flat-twisted tubing at a higher flow rate.
Finally, partition efficiency of the various column lengths of flattened zigzag tubing was compared. The capacity of each zigzag tubing layer is about 0.5 ml. The results show that Rs linearly increases with increased column capacity or column length (Fig. 6). When the capacity is about 0.25 ml (half a layer), two test samples still can be resolved at Rs = 1.05. Using the 1.5 layers of tubing with 0.8 ml capacity the Rs can reach to 1.48 which achieved complete separation of two test samples.
With lower column capacity the sample size can be reduced to obtain the excellent resolution. A series of experiments was performed to determine the optimum sample size using flattened tubing (Fig. 7). With increased sample size, peak resolution was sharply decreased. The 5 μl, sample size yielded the best separation at Rs = 1.17, whereas a large sample size of 40μl produced a single peak at Rs< 0.55.
In the zigzag toroidal column, plain and flattened tubing both have very good performance. The flattened tubing produces slightly higher peak resolution than the plain tubing and even with the decreased capacity at 0.25 ml, it yields the peak resolution of over Rs = 1. Especially the vert-flattened tubing can yield a high peak resolution with a short column in lower aqueous phase mobile which can be efficiently used for analytical separation of a minute amount of test samples for CCC/MS.