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COUNTERCURRENT CHROMATOGRAPHY | Solvent Extraction with a Helical Column
252 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column Conway WD (1990) Countercurrent Chromatography: Apparatus, Theory and Applications. New York: VCH. Foucault A (1994) Centrifugal Partition Chromatography. New York: Dekker. Ito Y (1981) Countercurrent chromatography. Journal of Biochemical and Biophysical Methods 5: 105–129. Ito Y (1984) Development of high-speed countercurrent chromatography. In: Giddings JC, Grushka E, Cazes J, and Brown PR (eds.) Advances in Chromatography, vol. 24, pp. 181–226. New York: Dekker. Ito Y (1986) High-speed countercurrent chromatography. CRC Critical Review in Analytical Chemistry 17: 65–143.
Ito Y (1991) Countercurrent chromatography in chromatography V. Journal of Chromatography Library, Part A, ch. 2. Amsterdam: Elsevier. Ito Y and Conway WD (1996) High-Speed Countercurrent Chromatography. New York: Wiley-Interscience. Ito Y and Ma Y (1996) pH-zone-refining countercurrent chromatography. Journal of Chromatography A 753: 1–36. Mandava NB and Ito Y (1988) Countercurrent Chromatography: Theory and Practice. New York: Dekker. Menet JM and Thie´baut D (1999) Countercurrent Chromatography. New York: Dekker.
Solvent Extraction with a Helical Column Y Ito, National Heart, Lung, and Blood Institute, Bethesda, MD, USA
Head
Air bubbles
Tail
& 2005, Elsevier Ltd. All Rights Reserved.
Water
Introduction Three steps required for multistage solvent extraction, i.e., phase mixing, phase settling, and transfer of the mobile phase, are defined clearly in the discontinuous countercurrent distribution process using the Craig apparatus. These basic requirements are essentially fulfilled by the use of a coiled tube in a continuous fashion. Solvent extraction using a coiled column is most efficiently performed with a horizontally laid coil that rotates about its own axis. In this horizontal coil orientation, the rotation induces the well known Archimedean screw force, which can be utilized for performing countercurrent solvent extraction.
(A)
Beads Head
Lower phase Head
This Archimedean screw effect on the hydrodynamic distribution of the solvent phases is illustrated in Figure 1, where each coil, consisting of five helical turns, is placed horizontally and rotated slowly around its axis. In Figure 1A, air bubbles and glass beads are introduced in the coil, previously filled with water, and both ends of the coil are sealed. Then, rotation of the coil in the indicated direction induces an Archimedean screw force that drives all the suspended objects (lighter or heavier than the
Upper phase Tail
(B) Head
Hydrodynamic Motion of Two Immiscible Solvent Phases in a Rotating Coil
Tail
Tail
(C) Figure 1 Archimedean screw effect in a rotating coil. (A) Motion of air bubbles and glass beads suspended in water; (B) Motion of droplets of one phase of an equilibrated two-phase solvent system suspended in the other phase; (C) Hydrodynamic equilibrium of two immiscible solvent phases in a slowly rotating coil.
water) toward the end of the coil labeled ‘head’ (the other side of the coil is called the ‘tail’). Under a slow coil rotation, the air bubbles (which are lighter than the water) always stay at the top of the coil, while the
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 253
glass beads (which are heavier than the water) stay at the bottom of the coil, both moving toward the head of the coil at a rate of one helical turn per rotation of the coil. Finally, both air bubbles and glass beads reach the head of the coil, where they remain by repeating a back and forth motion synchronous with the rotation of the coil. In Figure 1B, similar experiments are performed with a two-phase solvent system. The first coil is filled with the lighter phase (white) of an equilibrated two-phase solvent system, and a small amount of the heavier phase (black) is added. Then, under a slow rotation of the coil, droplets of the heavier phase remain at the bottom of the coil, traveling through the coil at a rate equal to the coil rotation. Similarly, the second coil is filled with the heavier phase (black), and a small amount of the lighter phase (white) is added. Under a slow rotation, droplets of the lighter phase stay at the top of the coil, again traveling toward the head of the coil at a rate of one turn per rotation of the coil. In Figure 1C, the coil is filled with nearly equal volumes of the two phases and rotated slowly about its axis. In this case, the lighter phase stays at the upper portion and the heavier phase at the lower portion of the coil, both competitively advancing toward the head of the coil. Sooner or later, the two phases establish a hydrodynamic equilibrium where each phase occupies about an equal space on the head side of the coil, and any excess of either phase remains at the tail end of the coil. Once this hydrodynamic equilibrium is formed, continued rotation of the coil mixes the two solvent phases vigorously, while the overall distribution of the two phases remains unaltered. This hydrodynamic equilibrium can be used for performing solute extraction in the following manner: The coil is first filled entirely with the stationary phase, either the lighter or the heavier phase, and the sample solution dissolved in either phase is introduced at the head side of the coil. Then, the mobile phase is eluted through the coil from the head toward the tail while the coil is slowly rotated around its axis. As the mobile phase meets the stationary phase in the rotating coil, the two solvent phases establish a hydrodynamic equilibrium quickly: the two phases are vigorously mixed by rotation of the coil, while some amount of the stationary phase is permanently retained in the coil. This process continues in each helical turn of the coil. After the entire coil attains a hydrodynamic equilibrium state and the mobile phase begins to elute from the tail end of the coil, the mobile phase only displaces the same phase in the coil, leaving the retained stationary phase permanently in the coil. Consequently, the solutes present
in the sample solution are subjected to an efficient partition process as in the multistage countercurrent distribution with the Craig apparatus but in a continuous manner. The partition efficiency in this partition system is highly dependent upon the amount of the stationary phase retained in the column. Under a slow rotation of the coil as described above, the two solvent phases occupy competitively the head side of the coil where the elution of either phase from the head of the coil only permits retention of the stationary phase at a maximum level of 50% of the column capacity. It has been found, however, that the volume ratio of the two phases occupying the head side of the coil can be altered by increasing the rotation speed of the coil. In Figure 2, three diagrams indicate the effect of rotation speed on the two-phase distribution on the head side of the coil. The experiments were performed using a set of coils with helical diameters of 3, 10, and 20 cm as indicated on the left. Each coil was first filled with about equal volumes of the lighter and heavier phases of chloroform/acetic acid/ 0.1 mol l 1 hydrochloric acid (2:2:1, v/v), closed at both ends, and then rotated at the desired speed. After a hydrodynamic phase equilibrium was reached, the rotation was stopped and the volume of the two phases occupying head side of the coil was measured. The percentage volume of the heavier (lower) phase occupying the head of the coil was then plotted against the rotation speed of the coil. The three diagrams obtained from different helical diameters show common features: In the slow rotation speed, between 0 rpm and 30 rpm, the two solvent phases distribute fairly evenly in the coil (stage I). When the rotation speed is increased, the heavier nonaqueous phase tends to occupy more space on the head side of the coil, and at a critical speed between 60 rpm and 100 rpm, the two phases are almost completely separated along the length of the coil, the heavier phase occupying the head side and the lighter phase the tail side of the coil (stage II). This particular two-phase distribution is called bilateral and most efficiently utilized for performing solvent extraction. After this critical speed range, the amount of heavier phase on the head side tends to decrease rather sharply, crossing below the 50% line (stage III). A further increase in the rotational speed again yields an even distribution of the two phases in the coil (stage IV). As the helical diameter increases, all these stages tend to shift toward the lower rpm range, apparently due to the enhanced centrifugal force field. Series of similar studies have been carried out using various two-phase solvent systems with a broad spectrum of hydrophobicity. Figure 3 illustrates a set
254 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column Phase distribution diagrams for coaxially rotated coil 100
Helical diameter 3 cm
50
0
50
0
100 0
100 10 cm
50
50
0
100 0
100
Distribution of upper phase (%)
10 cm
Distribution of lower phase (%)
3 cm
20 cm
20 cm
50
50
0 0
50
100
150
200 rpm
250
300
350
Figure 2 Hydrodynamic distribution of a two-phase solvent system composed of chloroform/acetic acid/0.1 mol l (2:2:1, v/v) in rotating coils of three different helical diameters, shown on the left.
of phase distribution diagrams obtained from nine commonly used volatile solvent systems in glass coils of various sizes with or without a silicone coating. These diagrams are arranged from left to right in the order of hydrophobicity of the major organic solvents as labeled at the top of each column, whereas the inner diameter (ID) and core diameter of the coils are indicated on the left margin. In each diagram the solid curve was obtained from an untreated coil and the broken curve from the same coil after silicone coating. Thus, any difference between these two curves indicates the effects of the solvent– wall interaction. An absence of one or both distribution curves in the designated space indicates that the two solvent phases failed to move or displayed sluggish motion in the coil and, therefore, the measurement could not be completed. These data indicate that with only a few exceptions, various two-phase solvent systems establish a bilateral hydrodynamic distribution in 1–2 cm ID coils at rotation speeds Bl00 rpm.
Mechanism of Countercurrent Extraction As mentioned above, the bilateral hydrodynamic distribution of the two phases can be utilized efficiently
400 1
100
hydrochloric acid
for performing solvent extraction. Figure 4 illustrates schematically the hydrodynamic mechanisms in solvent extraction using the basic hydrodynamic distribution (stage I) under a slow coil rotation (left) and the bilateral hydrodynamic distribution (stage II) at the critical rotation speed (right). For simplicity, all rotating coils – except for one shown at the top – are drawn uncoiled to show the overall distribution of the two solvent phases along the length of the coil. In the basic hydrodynamic equilibrium system (left), the slow rotation of the coil distributes the two phases evenly from the head of the coil (top). In order to obtain retention of the stationary phase in the coil under this hydrodynamic condition, the mobile phase, regardless of whether it is the heavier or lighter phase, should be introduced from the head of the coil. This operation results in a low level of stationary phase retention, usually much less than 50% of the total column capacity, as shown in the diagram. Elution of either phase from the tail of the coil would result in a total loss of the stationary phase from the coil. In the bilateral hydrodynamic equilibrium system (right), the critical rotation speed distributes the two phases bilaterally along the length of the coil, the head phase (white) entirely occupying the head side and the tail phase (gray) the tail side of the coil as
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 255 Two-phase distribution in rotating coils Hexane system
Ethyl acetate system
Hexane
Hexane
Ethyl acetate
Water
Methanol
Water
10
20
Heavier phase volume (%)
5
Chloroform 2 Acetic acid 2
Chloroform Water
4
Water
1
n-Butanol Water
n-Butanol 4 Acetic acid 1 Water 5
sec.-Butanol Water
50 0 100 50 0 100 50 0 100 50 0
5
10 20
Heavier phase volume (%)
Large-bore coil (∼20 mm I.D.)
Small-bore coil (∼10 mm I.D.)
Core diameter (cm) 100 2.5
Ethyl acetate 4 Acetic acid 1 Water
Butanol system
Chloroform system
100 50 0 100 50 0 100 50 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 RPM Untreated glass coil Silicone coated coil
Figure 3 Phase distribution diagrams for nine volatile two-phase solvent systems obtained from glass coils with various dimensions, as indicated on the left. The solid curve indicates the data obtained from nontreated glass coils and the dotted curve, from siliconetreated glass coils. The thin vertical line in each diagram indicates the rpm value at which the centrifugal force field created by the rotation equals unit gravity. Note that most solvent systems exhibit the critical rpm value where one phase occupies l00% of the column space on the head side of the coil.
al ic rit e C rat
Basic HDES
ro Slo ta w tio n
Principle of unilateral HDES
{
Tail
One helical turn Flow
Head
Tail
{
Head
Unilateral HDES
Hydrodynamic equilibrium
Hydrodynamic equilibrium
One helical turn Normal elution mode Reversed elution mode
Dual countercurrent system
Sample feed
Figure 4 Mechanism of solvent extraction with rotating coils. Left: basic hydrodynamic distribution produced by a slow coil rotation (stage I). Right: bilateral hydrodynamic distribution produced by the critical rotation speed (stage II).
shown in the first coil. In a rotating coil under unit gravity, the heavier phase usually becomes the head phase (see Figure 3). This hydrodynamic equilibrium condition indicates clearly that the tail phase, if
introduced at the head end of the coil, would travel through the head phase toward the tail and that the head phase, if introduced at the tail end of the coil, would travel through the tail phase toward the head.
256 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column
This hydrodynamic trend is effectively utilized for performing the solvent extraction in two different manners: The tail phase is eluted from the head toward the tail of the coil, previously filled with the head phase. Alternatively, the head phase is eluted from the tail toward the head of the coil, previously filled with the tail phase. In either case, the mobile phase can travel quickly through the coil, leaving a large volume of the stationary phase in the coil. This bilateral hydrodynamic system also permits simultaneous elution of the two solvent phases through the respective ends of the coil as shown at the bottom of the diagram. This dual countercurrent operation requires an additional flow tube at each end of the coil to collect the effluent and, if desired, a sample feed tube at the middle portion of the coil for continuous sample feeding. Comparison of the above two hydrodynamic systems reveals that the bilateral hydrodynamic system (Figure 4, right) provides several advantages over the basic hydrodynamic system (Figure 4, left): the bilateral system gives a better retention of the stationary phase in the column and yields a higher partition efficiency in a given period of time due to more efficient phase mixing under a higher rotation speed of the coil. The system can also be applied to dual countercurrent operation, where the two solvent phases literally undergo countercurrent movement through a coiled column.
Apparatus for Solvent Extraction Three different types extraction instrument based on the bilateral hydrodynamic equilibrium are used: a slow rotary countercurrent apparatus (Figure 5) and two types of high-speed centrifuge systems, one for standard extraction (Figure 6) and the other for dual countercurrent extraction, with a spiral column
(Figure 7). All these instruments are free of rotary seals. Slow Rotary Countercurrent Chromatography Apparatus
The slow rotary countercurrent chromatography (CCC) apparatus holds a long column holder, which slowly rotates about its axis (Figure 5). The separation column is prepared by winding a long Teflon tube directly onto the holder hub, making multiple coiled layers. Two types of Teflon tube were used, standard tubing and convoluted tubing (similar to a miniature vacuum cleaner duct). A pair of flow tubes from each terminal is passed through the hole of the hollow central shaft and then making an arch supported by a lateral tube support and then exiting the centrifuge system through another hole on the central shaft. The motor drives the rotary frame around the central axis of the centrifuge. Because the pulley at the right end of the countershaft is engaged to the stationary pulley with a toothed belt, the countershaft counter-rotates at the same rate on the rotating rotary frame. This motion is further conveyed to the column holder by a 1:1 gear coupling between the countershaft and the column holder. Consequently, the column holder rotates at the doubled speed with respect to the earth. This system allows the flow tubes to rotate without twisting, thus eliminating the need for rotary seals, and the high-speed centrifuge based on this rotary-seal-free system is widely used for apheresis at blood banks. Standard High-Speed CCC System
The multilayer coil assembly described above utilizes the Archimedean screw effect produced by the unit gravity. Thus, the relatively weak gravitational field limits the efficiency of the system. It has been found that the use of a centrifugal force field enhances the
2 5
4
7 6
3
1
Figure 5 Cross-sectional view of seal-free slow rotary countercurrent chromatography (CCC) equipped with a large multilayer coil.
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 257
Planetary gear
Column holder II = 0.5
Short coupling pipe
Toothed pulley Stationary gear Stationary pipe Flow tubes
Column holder I = 0.75
Planetary gear
Toothed pulley
Flow tubes
Toothed belt
Motor
Coiled column
Figure 6 Cross-sectional view of the coil planet centrifuge used for solvent extraction. One column holder (bottom) holds a coiled column, while the other (top) serves as a counterbalance.
partition efficiency in terms of both theoretical plate and the elution time. Among various coil planet centrifuge systems developed in the 1980s and 1990s, the type-J synchronous system produces a particular mode of planetary motion that yields a bilateral hydrodynamic equilibrium of two solvent phases in a multilayer coil mounted coaxially around the holder. Consequently, the system is applied efficiently to both solvent extraction and CCC. A cross-sectional view of the type-J coil planet centrifuge is illustrated in Figure 6. The motor (left,
bottom) drives the rotary frame via a pair of toothed pulleys and a toothed belt. The rotary frame holds a pair of column holders in symmetrical positions at a distance of 10 cm from the centrifuge axis. Each holder is equipped with a planetary gear that is engaged to an identical stationary sun gear (shaded) mounted rigidly around the central stationary pipe (shaded). This gear arrangement produces a planetary motion synchronous with the holder, i.e., one rotation about its own axis for each revolution around the central axis of the centrifuge in the same direction. A single-layer coiled column was mounted
258 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column Spiral disk for dual CCC (12.5 cm diameter, 1.2 cm thick, Kel-F) Separation channel: 1 mm wide and 2 mm deep with 2 mm ridge (3 mm pitch) O O
O O
O
O
O
O
O
O
O
O O
O
S O I2 O
O O1
O
O O2 O O I1
O
O-ring O
O
O
O
O
O
O O
O
Flow tubes O
O
I1: Inlet 1
O-ring O
O
O
I2: Inlet 2 O1: Outlet 1 O2: Outlet 2 S: Sample feed O2I1
I2
S
O1
(A) Clamps
Bearing block
Counter weight
Column Sun gear
Motor Planetary gear
Pulley
Flow tubes Toothed belt
(B) Figure 7 Instrumentation of dual countercurrent chromatograph. (A) Design of the spiral disk; (B) cross-sectional view of the apparatus.
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 259
Upper aqueous phase mobile
70
DNP-glu Absorbance (430 nm)
around one column holder (lower), and the other holder (upper) served to counterbalance the centrifuge system. A pair of flow tubes from the separation coil first passes through the center hole on the holder shaft and then, by making an arch, reaches the side hole made on the short coupling pipe to enter the opening of the central stationary pipe. As mentioned elsewhere these tubes are not twisted during a centrifuge run.
60 50 40
DNP-ala
30 20 10
Spiral Disk Dual Countercurrent Extraction Centrifuge
0
5 Time (h)
(A)
10
Lower nonaqueous phase mobile Absorbance (430 nm)
Figure 7 illustrates the new dual countercurrent extraction system (see Figure 4) using a spiral disk (A) mounted on the type-J synchronous planetary centrifuge system (B). The use of spiral channel configuration generates a radial centrifugal force gradient, enhancing the countercurrent movement of the two phases. This instrument is designed for separation of small amounts of pesticide in vegetable oil for mass spectrometric analysis. Because there is no stationary phase, multiple sample injections at regular intervals become possible without a risk of depleting the stationary phase or accumulating extracted oil, which would occur in the multilayer coil in the standard high-speed CCC technique.
0
DNP-ala
60 50 40 30 20
DNP-glu
10 0 0
(B)
5
10
15
Time (h)
Solvent Extraction with a Rotating Coil in a Unit Gravity
Figure 8 Separation of DNP amino acids with a multilayer coil rotating in a unit gravity. Column: multilayer coil, 30 m long, 5.5 mm ID FEP tube with capacity 750 ml. Sample: DNP-glu and DNP-ala, 500 mg of each dissolved in 30 ml of solvent. Solvent system: chloroform/glacial acetic acid/0.1 mol l 1 hydrochloric acid (2:2:1, v/v). Mobile phase: lighter aqueous phase (A) and heavier nonaqueous phase (B). Flow rate: 516 ml h 1. Coil rotation: 80 rpm.
Hydrodynamic studies (Figure 2) using the three coiled columns, with helical diameters ranging from 3 cm to 20 cm, demonstrated that the bilateral hydrodynamic distribution is established in all these coils B100 rpm. This result indicates that a long coiled column can be fabricated compactly by winding a single piece of plastic tubing around a spoolshaped rotary drum, making multiple coiled layers with dimensions as large as a 20 cm outside diameter (OD). The performance of this bilateral hydrodynamic extraction system in separating 2,4-dinitrophenyl (DNP) amino acids in a two-phase solvent system composed of chloroform, glacial acetic acid, and 0.1 mol l 1 hydrochloric acid at a volume ratio of 2:2:l was examined. A large capacity multilayer coil was fabricated from a 30 m long, 5.5 mm ID fluorinated ethylene propylene (FEP) tube by winding it coaxially onto a 10 cm diameter, 25 cm wide spool support, making three layers of the coil with a total capacity of B750 ml. Figure 8 shows the results of this preliminary separation, where both upper (A) and lower (B) phases were used as the mobile phase. In both separations two DNP amino acids are well resolved and eluted
out as symmetrical peaks. The partition efficiency, expressed in terms of the theoretical plate number, is over 200, which is nearly equivalent to that obtained from 200 partition units in the Craig countercurrent distribution apparatus. This method can be applied to extraction or separation of various compounds using suitable two-phase solvent systems. This slow rotary countercurrent extraction system has been applied for a large scale preparative separation of natural products using a multilayer coil prepared from convoluted PTFE tubing as shown in Figure 9. About 150 g amount of crude tea leaf extract was separated into four components in 72 h. In this separation, over 40 g of epigallocatechin gallate (EGCG) (fourth peak) was obtained at a high purity of 92.7%. The convoluted tubing provides some advantages over the standard tubing, such as ease of coil preparation and higher retention of the organic stationary phase. This low-speed rotary coil extraction system has various desirable features for industrial applications: the sample loading capacity can be scaled up simply by increasing the diameter of the multilayer coil
260 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column OH
OH OH
OH HO
HO
O
OH H H OH O OH H3C EGC O Unknown catechin 6500
N
OH CH3 N HO
OH O
N
9500
OH
OH
OH OH H H O C OH
O EGCG
OH
OH
CH3 Caffeine 8000
O
OH Epicatechin 11 000 12 500 14 000 15 500 17 000 18 500 20 000 21 500 Elution volume (ml)
Figure 9 Separation of crude extract of tea leaves using low-speed countercurrent extraction apparatus equipped with a multilayer coil of convoluted tubing. Column: multilayer coil made of convoluted PTFE tubing, 200 m long, 8.5 mm average ID coiled B9 cm OD holder hub, forming seven layers, each consisting of 60 loops with a total capacity of 10 l (see Figure 5). Sample: 150 g of tea leaf extract dissolved in 1.2 l of solvent consisting of equal volumes of each phase. Solvent system: n-hexane/ethyl acetate/n-butanol/ acetic acid/water (0.5:1:2:0.2:6, v/v). Mobile phase: lower aqueous phase. Elution mode: head to tail. Flow rate: 5 ml min 1. Column rotation: 21 rpm. Retention of the stationary phase: 33%.
and/or the width of the coil holder. The system also provides excellent safety features such as low rotation speed, low column pressure, and minimum risk of leakage of the solvent. Because of its simplicity, the system may be automated easily for long-term operation.
Solvent Extraction with a Coil Planet Centrifuge Extraction of DNP Amino Acids
Continuous countercurrent extraction is efficiently performed using the high-speed coil planet centrifuge as shown in Figure 6. It enables extraction of a solute present in a large volume of the mobile phase into a small volume of the stationary phase retained in the coiled column. This requires a set of conditions such that the solute must favor partition to the stationary phase. With commonly used extraction media such as an ethyl acetate–aqueous system, partition coefficients of various biological materials can be adjusted conveniently by modifying the pH and/or ionic strength of the aqueous phase to meet the above requirement. For the model studies, a pair of DNPamino acids, N-2,4-DNP-L-leucine (DNP-leu) and d-N-2,4-DNP-L-ornithine (DNP-orn), were selected as samples because they are readily observed through the column wall during the extraction process under stroboscopic illumination and also provide suitable partition coefficients. A typical extraction procedure may be divided into three steps, i.e., extraction, cleaning, and collection. In each operation, the column was filled with the stationary phase and the mobile phase containing the
sample was eluted through the column in the proper direction while the apparatus was run at 600 rpm. The extraction process was continued until 400 ml of the mobile phase was eluted. Then the mobile phase was replaced by the same phase but free of solute to wash the column contents. This cleaning process was continued until the additional 100 ml of the mobile phase was eluted. This would elute out all impurities having a partition coefficient of 0.1 or greater. The sample extracted into the stationary phase in the coiled column was collected by eluting with the mobile phase in the opposite direction. The sample still remaining in the column was then washed out by eluting the column with the other phase originally used as the stationary phase. The degree of sample recovery was estimated by comparing the amount of the sample in the original mobile phase with that in the collected stationary phase. The results of the experiments are summarized in Table 1. In experiments 1–3, DNP-leu dissolved in the aqueous mobile phase was extracted into B10 ml of the stationary nonaqueous phase. The sample recovery ranges from 94% to l00%. In experiments 4 and 5, DNP-orn dissolved in the nonaqueous mobile phase was extracted into the aqueous stationary phase. The sample recovery was in the range 97– 100%. In practice, application of the method to aqueous crude extracts or physiological fluids requires a preliminary adjustment of the solvent composition for providing a suitable partition coefficient of the desired material for the applied pair of solvents. In this case, pre-equilibration of the two phases may not be essential. Experiment 6 shows an example of operation with such nonequilibrated solvents. The sample DNP-leu was first dissolved in
99 6.1 600 (Head–tail) Partition coefficient is defined as solute concentration in the mobile phase divided by that in the stationary phase.
Extraction of Urinary Drug Metabolites
a
6
5
4
3
400 ml of 0.5 mol l 1 NaH2PO4 aqueous solution containing ethyl acetate at 5%, which is slightly below the saturation level of B7%. The column was filled with ethyl acetate followed by elution with the above sample solution. Both extraction and cleaning processes were performed as in other experiments. The sample solution collected from the column measured slightly over 6 ml. This depletion of the stationary phase resulted apparently from use of the nonequilibrated solvent pair but without any effect on the sample recovery. The overall results indicate the potential usefulness of the present method in processing large amounts of crude extracts or biological fluids in research laboratories. A small amount of the sample present in several hundred milliliters of the original solution can be enriched in 10 ml of the nonaqueous phase free of salt in 1 h, at a high recovery rate.
1
516 ml h 400 0.4 5% Ethylacetate in Ethylacetate DNP-leu 0.5 mol NaH2PO4 (o0.01)
100 11.8 600 (Tail–head) 1
516 ml h 400 0.04 (o0.01) Aqueous Nonaqueous
97 11.8 600 (Tail–head) 1
516 ml h 400 0.4 DNP-orn (o0.01) Aqueous Nonaqueous
100 10.4 600 (Head–tail) 1
516 ml h 400 0.04 Nonaqueous Aqueous
(o0.01)
97 10.0 600 (Head–tail) 1
516 ml h 400 0.4 (o0.01) Nonaqueous 2
Ethylacetate 1 0.5 mol l 1 NaH2PO4 2 0.5 mol l 1 NaH2PO4 2 0.5 mol l 1 NaH2PO4 2 Ethylacetate 2 0.5 mol l 1 NaH2PO4 1 0.5 mol l 1 NaH2PO4 1 Nonequilibrium system 1
Aqueous
94 10.5 600 (Head–tail) 1
516 ml h 400 4 Nonaqueous DNP-leu (o0.01) Aqueous
Solvent system Exp. no.
Table 1 Experimental conditions and results of extraction
Sample (P.C.) a Stationary phase Mobile phase
Sample conc. Extracted Flow rate (direction) in mobile mobile phase phase volume (ml) (mg%)
RPM
Sample Collected recovery stationary phase volume (%) (ml)
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 261
The present method has been applied to extraction of urinary metabolites of daunorubicin, an anticancer drug. The extraction was performed with a twophase solvent system composed of n-butanol/ 0.3 mol l 1 Na2HPO4. Prior to the extraction, the urine sample was saturated with n-butanol, and then Na2HPO4 was added at a concentration of 0.3 mol l 1. In each experiment, the column was first filled with n-butanol. Then, the apparatus was rotated at 650 rpm while aqueous Na2HPO4 saturated with n-butanol was pumped into the column to equilibrate the stationary phase. The prepared urine sample, 1–2 l in volume, was then eluted through the column at a flow rate of 500–700 ml h 1 with a metering pump. After all the sample solution was eluted, the column was cleaned by eluting with 100 ml of aqueous, n-butanol-saturated 0.3 mol l 1 Na2HPO4. Then, the centrifuge run was terminated and the retained n-butanol phase was drained from the column by connecting the column inlet to an N2 line under pressure. Several milliliters of n-butanol were flushed through the column to recover any remaining sample. The n-butanol extracts were combined and evaporated to dryness by flash evaporation. The results of the experiments are illustrated in Figure 10, where the high-performance liquid chromatography (HPLC) analysis of the original urine sample and that of the countercurrent extract are compared. In Figure 10A, the chromatogram of the original urine sample shows a large amount of hydrophilic material at the solvent front and two metabolites peaks, D1 and D2. As shown in Figure 10B, the chromatogram of the countercurrent extract with the coil planet centrifuge reveals enriched D1 and D2
262 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column See also: Countercurrent Chromatography: Overview. Original urine Fluorescence
Further Reading
D2
0
2
4
(A)
D1
6 8 10 Retention time (min)
14
D2 D1 dD3
Fluorescence
Countercurrent extract
12
demethyl-dD3
4-O -Glucuronide
0
2
(B)
4 6 8 10 Retention time (min)
12
14
Figure 10 Extraction of urinary metabolites of daunorubicin using the coil planet centrifuge. (A) HPLC analysis of the original urine; (B) HPLC analysis of the countercurrent extract.
peaks and three additional metabolite peaks, dD3, demethyl-dD3, and 4-O-glucuronide as indicated in the chromatogram.
Ito Y (1981) New continuous extraction method with a coil planet centrifuge. Journal of Chromatography 207: 161–169. Ito Y and Bhatnagar R (1981) Preparative countercurrent chromatography with a rotating coil assembly. Journal of Chromatography 207: 171–180. Nakazawa H, Riggs CE Jr., Egorin MJ, et al. (1984) Extraction of urinary anthracycline antitumor antibiotics with the coil planet centrifuge. Journal of Chromatography 307: 323–333. Ito Y (1988) Studies on hydrodynamic distribution of two immiscible solvent phases in rotating coils. Journal of Liquid Chromatography 11: 1–19. Ito Y and Conway WD (eds.) (1996) High-Speed Countercurrent Chromatography. New York: Wiley Interscience. Menet JM and Thibaut D (eds.) (1999) Countercurrent Chromatography. New York: Dekker. Du Q-Z, Wu P-D, and Ito Y (2000) Low-speed rotary countercurrent chromatography using a convoluted multilayer helical tube for industrial separation. Analytical Chemistry 72: 3363–3365. Berthod (ed.) (2002) Countercurrent Chromatography: The Support-Free Liquid Stationary Phase. Amsterdam: Elsevier. Du Q-Z and Ito Y (2003) Slow rotary countercurrent chromatography. Journal of Liquid Chromatography and Related Technologies 26(11): 1827–1838. Du Q-Z, Winterhalter P, and Ito Y (2003) Large convoluted tubing for scale-up of slow rotary countercurrent chromatography. Journal of Liquid Chromatography and Related Technologies 26(12): 1991–2002. Ito Y (2003) Spiral Disk for Dual Countercurrent Extraction. US Patent pending.
CRMs See QUALITY ASSURANCE: Reference Materials; Production of Reference Materials
CSV See VOLTAMMETRY: Cathodic Stripping
CYCLIC VOLTAMMETRY See VOLTAMMETRY: Linear Sweep and Cyclic
Ito Y (1991) Countercurrent chromatography in chromatography V. Journal of Chromatography Library, Part A, ch. 2. Amsterdam: Elsevier. Ito Y and Conway WD (1996) High-Speed Countercurrent Chromatography. New York: Wiley-Interscience. Ito Y and Ma Y (1996) pH-zone-refining countercurrent chromatography. Journal of Chromatography A 753: 1–36. Mandava NB and Ito Y (1988) Countercurrent Chromatography: Theory and Practice. New York: Dekker. Menet JM and Thie´baut D (1999) Countercurrent Chromatography. New York: Dekker.
Solvent Extraction with a Helical Column Y Ito, National Heart, Lung, and Blood Institute, Bethesda, MD, USA
Head
Air bubbles
Tail
& 2005, Elsevier Ltd. All Rights Reserved.
Water
Introduction Three steps required for multistage solvent extraction, i.e., phase mixing, phase settling, and transfer of the mobile phase, are defined clearly in the discontinuous countercurrent distribution process using the Craig apparatus. These basic requirements are essentially fulfilled by the use of a coiled tube in a continuous fashion. Solvent extraction using a coiled column is most efficiently performed with a horizontally laid coil that rotates about its own axis. In this horizontal coil orientation, the rotation induces the well known Archimedean screw force, which can be utilized for performing countercurrent solvent extraction.
(A)
Beads Head
Lower phase Head
This Archimedean screw effect on the hydrodynamic distribution of the solvent phases is illustrated in Figure 1, where each coil, consisting of five helical turns, is placed horizontally and rotated slowly around its axis. In Figure 1A, air bubbles and glass beads are introduced in the coil, previously filled with water, and both ends of the coil are sealed. Then, rotation of the coil in the indicated direction induces an Archimedean screw force that drives all the suspended objects (lighter or heavier than the
Upper phase Tail
(B) Head
Hydrodynamic Motion of Two Immiscible Solvent Phases in a Rotating Coil
Tail
Tail
(C) Figure 1 Archimedean screw effect in a rotating coil. (A) Motion of air bubbles and glass beads suspended in water; (B) Motion of droplets of one phase of an equilibrated two-phase solvent system suspended in the other phase; (C) Hydrodynamic equilibrium of two immiscible solvent phases in a slowly rotating coil.
water) toward the end of the coil labeled ‘head’ (the other side of the coil is called the ‘tail’). Under a slow coil rotation, the air bubbles (which are lighter than the water) always stay at the top of the coil, while the
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 253
glass beads (which are heavier than the water) stay at the bottom of the coil, both moving toward the head of the coil at a rate of one helical turn per rotation of the coil. Finally, both air bubbles and glass beads reach the head of the coil, where they remain by repeating a back and forth motion synchronous with the rotation of the coil. In Figure 1B, similar experiments are performed with a two-phase solvent system. The first coil is filled with the lighter phase (white) of an equilibrated two-phase solvent system, and a small amount of the heavier phase (black) is added. Then, under a slow rotation of the coil, droplets of the heavier phase remain at the bottom of the coil, traveling through the coil at a rate equal to the coil rotation. Similarly, the second coil is filled with the heavier phase (black), and a small amount of the lighter phase (white) is added. Under a slow rotation, droplets of the lighter phase stay at the top of the coil, again traveling toward the head of the coil at a rate of one turn per rotation of the coil. In Figure 1C, the coil is filled with nearly equal volumes of the two phases and rotated slowly about its axis. In this case, the lighter phase stays at the upper portion and the heavier phase at the lower portion of the coil, both competitively advancing toward the head of the coil. Sooner or later, the two phases establish a hydrodynamic equilibrium where each phase occupies about an equal space on the head side of the coil, and any excess of either phase remains at the tail end of the coil. Once this hydrodynamic equilibrium is formed, continued rotation of the coil mixes the two solvent phases vigorously, while the overall distribution of the two phases remains unaltered. This hydrodynamic equilibrium can be used for performing solute extraction in the following manner: The coil is first filled entirely with the stationary phase, either the lighter or the heavier phase, and the sample solution dissolved in either phase is introduced at the head side of the coil. Then, the mobile phase is eluted through the coil from the head toward the tail while the coil is slowly rotated around its axis. As the mobile phase meets the stationary phase in the rotating coil, the two solvent phases establish a hydrodynamic equilibrium quickly: the two phases are vigorously mixed by rotation of the coil, while some amount of the stationary phase is permanently retained in the coil. This process continues in each helical turn of the coil. After the entire coil attains a hydrodynamic equilibrium state and the mobile phase begins to elute from the tail end of the coil, the mobile phase only displaces the same phase in the coil, leaving the retained stationary phase permanently in the coil. Consequently, the solutes present
in the sample solution are subjected to an efficient partition process as in the multistage countercurrent distribution with the Craig apparatus but in a continuous manner. The partition efficiency in this partition system is highly dependent upon the amount of the stationary phase retained in the column. Under a slow rotation of the coil as described above, the two solvent phases occupy competitively the head side of the coil where the elution of either phase from the head of the coil only permits retention of the stationary phase at a maximum level of 50% of the column capacity. It has been found, however, that the volume ratio of the two phases occupying the head side of the coil can be altered by increasing the rotation speed of the coil. In Figure 2, three diagrams indicate the effect of rotation speed on the two-phase distribution on the head side of the coil. The experiments were performed using a set of coils with helical diameters of 3, 10, and 20 cm as indicated on the left. Each coil was first filled with about equal volumes of the lighter and heavier phases of chloroform/acetic acid/ 0.1 mol l 1 hydrochloric acid (2:2:1, v/v), closed at both ends, and then rotated at the desired speed. After a hydrodynamic phase equilibrium was reached, the rotation was stopped and the volume of the two phases occupying head side of the coil was measured. The percentage volume of the heavier (lower) phase occupying the head of the coil was then plotted against the rotation speed of the coil. The three diagrams obtained from different helical diameters show common features: In the slow rotation speed, between 0 rpm and 30 rpm, the two solvent phases distribute fairly evenly in the coil (stage I). When the rotation speed is increased, the heavier nonaqueous phase tends to occupy more space on the head side of the coil, and at a critical speed between 60 rpm and 100 rpm, the two phases are almost completely separated along the length of the coil, the heavier phase occupying the head side and the lighter phase the tail side of the coil (stage II). This particular two-phase distribution is called bilateral and most efficiently utilized for performing solvent extraction. After this critical speed range, the amount of heavier phase on the head side tends to decrease rather sharply, crossing below the 50% line (stage III). A further increase in the rotational speed again yields an even distribution of the two phases in the coil (stage IV). As the helical diameter increases, all these stages tend to shift toward the lower rpm range, apparently due to the enhanced centrifugal force field. Series of similar studies have been carried out using various two-phase solvent systems with a broad spectrum of hydrophobicity. Figure 3 illustrates a set
254 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column Phase distribution diagrams for coaxially rotated coil 100
Helical diameter 3 cm
50
0
50
0
100 0
100 10 cm
50
50
0
100 0
100
Distribution of upper phase (%)
10 cm
Distribution of lower phase (%)
3 cm
20 cm
20 cm
50
50
0 0
50
100
150
200 rpm
250
300
350
Figure 2 Hydrodynamic distribution of a two-phase solvent system composed of chloroform/acetic acid/0.1 mol l (2:2:1, v/v) in rotating coils of three different helical diameters, shown on the left.
of phase distribution diagrams obtained from nine commonly used volatile solvent systems in glass coils of various sizes with or without a silicone coating. These diagrams are arranged from left to right in the order of hydrophobicity of the major organic solvents as labeled at the top of each column, whereas the inner diameter (ID) and core diameter of the coils are indicated on the left margin. In each diagram the solid curve was obtained from an untreated coil and the broken curve from the same coil after silicone coating. Thus, any difference between these two curves indicates the effects of the solvent– wall interaction. An absence of one or both distribution curves in the designated space indicates that the two solvent phases failed to move or displayed sluggish motion in the coil and, therefore, the measurement could not be completed. These data indicate that with only a few exceptions, various two-phase solvent systems establish a bilateral hydrodynamic distribution in 1–2 cm ID coils at rotation speeds Bl00 rpm.
Mechanism of Countercurrent Extraction As mentioned above, the bilateral hydrodynamic distribution of the two phases can be utilized efficiently
400 1
100
hydrochloric acid
for performing solvent extraction. Figure 4 illustrates schematically the hydrodynamic mechanisms in solvent extraction using the basic hydrodynamic distribution (stage I) under a slow coil rotation (left) and the bilateral hydrodynamic distribution (stage II) at the critical rotation speed (right). For simplicity, all rotating coils – except for one shown at the top – are drawn uncoiled to show the overall distribution of the two solvent phases along the length of the coil. In the basic hydrodynamic equilibrium system (left), the slow rotation of the coil distributes the two phases evenly from the head of the coil (top). In order to obtain retention of the stationary phase in the coil under this hydrodynamic condition, the mobile phase, regardless of whether it is the heavier or lighter phase, should be introduced from the head of the coil. This operation results in a low level of stationary phase retention, usually much less than 50% of the total column capacity, as shown in the diagram. Elution of either phase from the tail of the coil would result in a total loss of the stationary phase from the coil. In the bilateral hydrodynamic equilibrium system (right), the critical rotation speed distributes the two phases bilaterally along the length of the coil, the head phase (white) entirely occupying the head side and the tail phase (gray) the tail side of the coil as
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 255 Two-phase distribution in rotating coils Hexane system
Ethyl acetate system
Hexane
Hexane
Ethyl acetate
Water
Methanol
Water
10
20
Heavier phase volume (%)
5
Chloroform 2 Acetic acid 2
Chloroform Water
4
Water
1
n-Butanol Water
n-Butanol 4 Acetic acid 1 Water 5
sec.-Butanol Water
50 0 100 50 0 100 50 0 100 50 0
5
10 20
Heavier phase volume (%)
Large-bore coil (∼20 mm I.D.)
Small-bore coil (∼10 mm I.D.)
Core diameter (cm) 100 2.5
Ethyl acetate 4 Acetic acid 1 Water
Butanol system
Chloroform system
100 50 0 100 50 0 100 50 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 RPM Untreated glass coil Silicone coated coil
Figure 3 Phase distribution diagrams for nine volatile two-phase solvent systems obtained from glass coils with various dimensions, as indicated on the left. The solid curve indicates the data obtained from nontreated glass coils and the dotted curve, from siliconetreated glass coils. The thin vertical line in each diagram indicates the rpm value at which the centrifugal force field created by the rotation equals unit gravity. Note that most solvent systems exhibit the critical rpm value where one phase occupies l00% of the column space on the head side of the coil.
al ic rit e C rat
Basic HDES
ro Slo ta w tio n
Principle of unilateral HDES
{
Tail
One helical turn Flow
Head
Tail
{
Head
Unilateral HDES
Hydrodynamic equilibrium
Hydrodynamic equilibrium
One helical turn Normal elution mode Reversed elution mode
Dual countercurrent system
Sample feed
Figure 4 Mechanism of solvent extraction with rotating coils. Left: basic hydrodynamic distribution produced by a slow coil rotation (stage I). Right: bilateral hydrodynamic distribution produced by the critical rotation speed (stage II).
shown in the first coil. In a rotating coil under unit gravity, the heavier phase usually becomes the head phase (see Figure 3). This hydrodynamic equilibrium condition indicates clearly that the tail phase, if
introduced at the head end of the coil, would travel through the head phase toward the tail and that the head phase, if introduced at the tail end of the coil, would travel through the tail phase toward the head.
256 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column
This hydrodynamic trend is effectively utilized for performing the solvent extraction in two different manners: The tail phase is eluted from the head toward the tail of the coil, previously filled with the head phase. Alternatively, the head phase is eluted from the tail toward the head of the coil, previously filled with the tail phase. In either case, the mobile phase can travel quickly through the coil, leaving a large volume of the stationary phase in the coil. This bilateral hydrodynamic system also permits simultaneous elution of the two solvent phases through the respective ends of the coil as shown at the bottom of the diagram. This dual countercurrent operation requires an additional flow tube at each end of the coil to collect the effluent and, if desired, a sample feed tube at the middle portion of the coil for continuous sample feeding. Comparison of the above two hydrodynamic systems reveals that the bilateral hydrodynamic system (Figure 4, right) provides several advantages over the basic hydrodynamic system (Figure 4, left): the bilateral system gives a better retention of the stationary phase in the column and yields a higher partition efficiency in a given period of time due to more efficient phase mixing under a higher rotation speed of the coil. The system can also be applied to dual countercurrent operation, where the two solvent phases literally undergo countercurrent movement through a coiled column.
Apparatus for Solvent Extraction Three different types extraction instrument based on the bilateral hydrodynamic equilibrium are used: a slow rotary countercurrent apparatus (Figure 5) and two types of high-speed centrifuge systems, one for standard extraction (Figure 6) and the other for dual countercurrent extraction, with a spiral column
(Figure 7). All these instruments are free of rotary seals. Slow Rotary Countercurrent Chromatography Apparatus
The slow rotary countercurrent chromatography (CCC) apparatus holds a long column holder, which slowly rotates about its axis (Figure 5). The separation column is prepared by winding a long Teflon tube directly onto the holder hub, making multiple coiled layers. Two types of Teflon tube were used, standard tubing and convoluted tubing (similar to a miniature vacuum cleaner duct). A pair of flow tubes from each terminal is passed through the hole of the hollow central shaft and then making an arch supported by a lateral tube support and then exiting the centrifuge system through another hole on the central shaft. The motor drives the rotary frame around the central axis of the centrifuge. Because the pulley at the right end of the countershaft is engaged to the stationary pulley with a toothed belt, the countershaft counter-rotates at the same rate on the rotating rotary frame. This motion is further conveyed to the column holder by a 1:1 gear coupling between the countershaft and the column holder. Consequently, the column holder rotates at the doubled speed with respect to the earth. This system allows the flow tubes to rotate without twisting, thus eliminating the need for rotary seals, and the high-speed centrifuge based on this rotary-seal-free system is widely used for apheresis at blood banks. Standard High-Speed CCC System
The multilayer coil assembly described above utilizes the Archimedean screw effect produced by the unit gravity. Thus, the relatively weak gravitational field limits the efficiency of the system. It has been found that the use of a centrifugal force field enhances the
2 5
4
7 6
3
1
Figure 5 Cross-sectional view of seal-free slow rotary countercurrent chromatography (CCC) equipped with a large multilayer coil.
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 257
Planetary gear
Column holder II = 0.5
Short coupling pipe
Toothed pulley Stationary gear Stationary pipe Flow tubes
Column holder I = 0.75
Planetary gear
Toothed pulley
Flow tubes
Toothed belt
Motor
Coiled column
Figure 6 Cross-sectional view of the coil planet centrifuge used for solvent extraction. One column holder (bottom) holds a coiled column, while the other (top) serves as a counterbalance.
partition efficiency in terms of both theoretical plate and the elution time. Among various coil planet centrifuge systems developed in the 1980s and 1990s, the type-J synchronous system produces a particular mode of planetary motion that yields a bilateral hydrodynamic equilibrium of two solvent phases in a multilayer coil mounted coaxially around the holder. Consequently, the system is applied efficiently to both solvent extraction and CCC. A cross-sectional view of the type-J coil planet centrifuge is illustrated in Figure 6. The motor (left,
bottom) drives the rotary frame via a pair of toothed pulleys and a toothed belt. The rotary frame holds a pair of column holders in symmetrical positions at a distance of 10 cm from the centrifuge axis. Each holder is equipped with a planetary gear that is engaged to an identical stationary sun gear (shaded) mounted rigidly around the central stationary pipe (shaded). This gear arrangement produces a planetary motion synchronous with the holder, i.e., one rotation about its own axis for each revolution around the central axis of the centrifuge in the same direction. A single-layer coiled column was mounted
258 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column Spiral disk for dual CCC (12.5 cm diameter, 1.2 cm thick, Kel-F) Separation channel: 1 mm wide and 2 mm deep with 2 mm ridge (3 mm pitch) O O
O O
O
O
O
O
O
O
O
O O
O
S O I2 O
O O1
O
O O2 O O I1
O
O-ring O
O
O
O
O
O
O O
O
Flow tubes O
O
I1: Inlet 1
O-ring O
O
O
I2: Inlet 2 O1: Outlet 1 O2: Outlet 2 S: Sample feed O2I1
I2
S
O1
(A) Clamps
Bearing block
Counter weight
Column Sun gear
Motor Planetary gear
Pulley
Flow tubes Toothed belt
(B) Figure 7 Instrumentation of dual countercurrent chromatograph. (A) Design of the spiral disk; (B) cross-sectional view of the apparatus.
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 259
Upper aqueous phase mobile
70
DNP-glu Absorbance (430 nm)
around one column holder (lower), and the other holder (upper) served to counterbalance the centrifuge system. A pair of flow tubes from the separation coil first passes through the center hole on the holder shaft and then, by making an arch, reaches the side hole made on the short coupling pipe to enter the opening of the central stationary pipe. As mentioned elsewhere these tubes are not twisted during a centrifuge run.
60 50 40
DNP-ala
30 20 10
Spiral Disk Dual Countercurrent Extraction Centrifuge
0
5 Time (h)
(A)
10
Lower nonaqueous phase mobile Absorbance (430 nm)
Figure 7 illustrates the new dual countercurrent extraction system (see Figure 4) using a spiral disk (A) mounted on the type-J synchronous planetary centrifuge system (B). The use of spiral channel configuration generates a radial centrifugal force gradient, enhancing the countercurrent movement of the two phases. This instrument is designed for separation of small amounts of pesticide in vegetable oil for mass spectrometric analysis. Because there is no stationary phase, multiple sample injections at regular intervals become possible without a risk of depleting the stationary phase or accumulating extracted oil, which would occur in the multilayer coil in the standard high-speed CCC technique.
0
DNP-ala
60 50 40 30 20
DNP-glu
10 0 0
(B)
5
10
15
Time (h)
Solvent Extraction with a Rotating Coil in a Unit Gravity
Figure 8 Separation of DNP amino acids with a multilayer coil rotating in a unit gravity. Column: multilayer coil, 30 m long, 5.5 mm ID FEP tube with capacity 750 ml. Sample: DNP-glu and DNP-ala, 500 mg of each dissolved in 30 ml of solvent. Solvent system: chloroform/glacial acetic acid/0.1 mol l 1 hydrochloric acid (2:2:1, v/v). Mobile phase: lighter aqueous phase (A) and heavier nonaqueous phase (B). Flow rate: 516 ml h 1. Coil rotation: 80 rpm.
Hydrodynamic studies (Figure 2) using the three coiled columns, with helical diameters ranging from 3 cm to 20 cm, demonstrated that the bilateral hydrodynamic distribution is established in all these coils B100 rpm. This result indicates that a long coiled column can be fabricated compactly by winding a single piece of plastic tubing around a spoolshaped rotary drum, making multiple coiled layers with dimensions as large as a 20 cm outside diameter (OD). The performance of this bilateral hydrodynamic extraction system in separating 2,4-dinitrophenyl (DNP) amino acids in a two-phase solvent system composed of chloroform, glacial acetic acid, and 0.1 mol l 1 hydrochloric acid at a volume ratio of 2:2:l was examined. A large capacity multilayer coil was fabricated from a 30 m long, 5.5 mm ID fluorinated ethylene propylene (FEP) tube by winding it coaxially onto a 10 cm diameter, 25 cm wide spool support, making three layers of the coil with a total capacity of B750 ml. Figure 8 shows the results of this preliminary separation, where both upper (A) and lower (B) phases were used as the mobile phase. In both separations two DNP amino acids are well resolved and eluted
out as symmetrical peaks. The partition efficiency, expressed in terms of the theoretical plate number, is over 200, which is nearly equivalent to that obtained from 200 partition units in the Craig countercurrent distribution apparatus. This method can be applied to extraction or separation of various compounds using suitable two-phase solvent systems. This slow rotary countercurrent extraction system has been applied for a large scale preparative separation of natural products using a multilayer coil prepared from convoluted PTFE tubing as shown in Figure 9. About 150 g amount of crude tea leaf extract was separated into four components in 72 h. In this separation, over 40 g of epigallocatechin gallate (EGCG) (fourth peak) was obtained at a high purity of 92.7%. The convoluted tubing provides some advantages over the standard tubing, such as ease of coil preparation and higher retention of the organic stationary phase. This low-speed rotary coil extraction system has various desirable features for industrial applications: the sample loading capacity can be scaled up simply by increasing the diameter of the multilayer coil
260 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column OH
OH OH
OH HO
HO
O
OH H H OH O OH H3C EGC O Unknown catechin 6500
N
OH CH3 N HO
OH O
N
9500
OH
OH
OH OH H H O C OH
O EGCG
OH
OH
CH3 Caffeine 8000
O
OH Epicatechin 11 000 12 500 14 000 15 500 17 000 18 500 20 000 21 500 Elution volume (ml)
Figure 9 Separation of crude extract of tea leaves using low-speed countercurrent extraction apparatus equipped with a multilayer coil of convoluted tubing. Column: multilayer coil made of convoluted PTFE tubing, 200 m long, 8.5 mm average ID coiled B9 cm OD holder hub, forming seven layers, each consisting of 60 loops with a total capacity of 10 l (see Figure 5). Sample: 150 g of tea leaf extract dissolved in 1.2 l of solvent consisting of equal volumes of each phase. Solvent system: n-hexane/ethyl acetate/n-butanol/ acetic acid/water (0.5:1:2:0.2:6, v/v). Mobile phase: lower aqueous phase. Elution mode: head to tail. Flow rate: 5 ml min 1. Column rotation: 21 rpm. Retention of the stationary phase: 33%.
and/or the width of the coil holder. The system also provides excellent safety features such as low rotation speed, low column pressure, and minimum risk of leakage of the solvent. Because of its simplicity, the system may be automated easily for long-term operation.
Solvent Extraction with a Coil Planet Centrifuge Extraction of DNP Amino Acids
Continuous countercurrent extraction is efficiently performed using the high-speed coil planet centrifuge as shown in Figure 6. It enables extraction of a solute present in a large volume of the mobile phase into a small volume of the stationary phase retained in the coiled column. This requires a set of conditions such that the solute must favor partition to the stationary phase. With commonly used extraction media such as an ethyl acetate–aqueous system, partition coefficients of various biological materials can be adjusted conveniently by modifying the pH and/or ionic strength of the aqueous phase to meet the above requirement. For the model studies, a pair of DNPamino acids, N-2,4-DNP-L-leucine (DNP-leu) and d-N-2,4-DNP-L-ornithine (DNP-orn), were selected as samples because they are readily observed through the column wall during the extraction process under stroboscopic illumination and also provide suitable partition coefficients. A typical extraction procedure may be divided into three steps, i.e., extraction, cleaning, and collection. In each operation, the column was filled with the stationary phase and the mobile phase containing the
sample was eluted through the column in the proper direction while the apparatus was run at 600 rpm. The extraction process was continued until 400 ml of the mobile phase was eluted. Then the mobile phase was replaced by the same phase but free of solute to wash the column contents. This cleaning process was continued until the additional 100 ml of the mobile phase was eluted. This would elute out all impurities having a partition coefficient of 0.1 or greater. The sample extracted into the stationary phase in the coiled column was collected by eluting with the mobile phase in the opposite direction. The sample still remaining in the column was then washed out by eluting the column with the other phase originally used as the stationary phase. The degree of sample recovery was estimated by comparing the amount of the sample in the original mobile phase with that in the collected stationary phase. The results of the experiments are summarized in Table 1. In experiments 1–3, DNP-leu dissolved in the aqueous mobile phase was extracted into B10 ml of the stationary nonaqueous phase. The sample recovery ranges from 94% to l00%. In experiments 4 and 5, DNP-orn dissolved in the nonaqueous mobile phase was extracted into the aqueous stationary phase. The sample recovery was in the range 97– 100%. In practice, application of the method to aqueous crude extracts or physiological fluids requires a preliminary adjustment of the solvent composition for providing a suitable partition coefficient of the desired material for the applied pair of solvents. In this case, pre-equilibration of the two phases may not be essential. Experiment 6 shows an example of operation with such nonequilibrated solvents. The sample DNP-leu was first dissolved in
99 6.1 600 (Head–tail) Partition coefficient is defined as solute concentration in the mobile phase divided by that in the stationary phase.
Extraction of Urinary Drug Metabolites
a
6
5
4
3
400 ml of 0.5 mol l 1 NaH2PO4 aqueous solution containing ethyl acetate at 5%, which is slightly below the saturation level of B7%. The column was filled with ethyl acetate followed by elution with the above sample solution. Both extraction and cleaning processes were performed as in other experiments. The sample solution collected from the column measured slightly over 6 ml. This depletion of the stationary phase resulted apparently from use of the nonequilibrated solvent pair but without any effect on the sample recovery. The overall results indicate the potential usefulness of the present method in processing large amounts of crude extracts or biological fluids in research laboratories. A small amount of the sample present in several hundred milliliters of the original solution can be enriched in 10 ml of the nonaqueous phase free of salt in 1 h, at a high recovery rate.
1
516 ml h 400 0.4 5% Ethylacetate in Ethylacetate DNP-leu 0.5 mol NaH2PO4 (o0.01)
100 11.8 600 (Tail–head) 1
516 ml h 400 0.04 (o0.01) Aqueous Nonaqueous
97 11.8 600 (Tail–head) 1
516 ml h 400 0.4 DNP-orn (o0.01) Aqueous Nonaqueous
100 10.4 600 (Head–tail) 1
516 ml h 400 0.04 Nonaqueous Aqueous
(o0.01)
97 10.0 600 (Head–tail) 1
516 ml h 400 0.4 (o0.01) Nonaqueous 2
Ethylacetate 1 0.5 mol l 1 NaH2PO4 2 0.5 mol l 1 NaH2PO4 2 0.5 mol l 1 NaH2PO4 2 Ethylacetate 2 0.5 mol l 1 NaH2PO4 1 0.5 mol l 1 NaH2PO4 1 Nonequilibrium system 1
Aqueous
94 10.5 600 (Head–tail) 1
516 ml h 400 4 Nonaqueous DNP-leu (o0.01) Aqueous
Solvent system Exp. no.
Table 1 Experimental conditions and results of extraction
Sample (P.C.) a Stationary phase Mobile phase
Sample conc. Extracted Flow rate (direction) in mobile mobile phase phase volume (ml) (mg%)
RPM
Sample Collected recovery stationary phase volume (%) (ml)
COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column 261
The present method has been applied to extraction of urinary metabolites of daunorubicin, an anticancer drug. The extraction was performed with a twophase solvent system composed of n-butanol/ 0.3 mol l 1 Na2HPO4. Prior to the extraction, the urine sample was saturated with n-butanol, and then Na2HPO4 was added at a concentration of 0.3 mol l 1. In each experiment, the column was first filled with n-butanol. Then, the apparatus was rotated at 650 rpm while aqueous Na2HPO4 saturated with n-butanol was pumped into the column to equilibrate the stationary phase. The prepared urine sample, 1–2 l in volume, was then eluted through the column at a flow rate of 500–700 ml h 1 with a metering pump. After all the sample solution was eluted, the column was cleaned by eluting with 100 ml of aqueous, n-butanol-saturated 0.3 mol l 1 Na2HPO4. Then, the centrifuge run was terminated and the retained n-butanol phase was drained from the column by connecting the column inlet to an N2 line under pressure. Several milliliters of n-butanol were flushed through the column to recover any remaining sample. The n-butanol extracts were combined and evaporated to dryness by flash evaporation. The results of the experiments are illustrated in Figure 10, where the high-performance liquid chromatography (HPLC) analysis of the original urine sample and that of the countercurrent extract are compared. In Figure 10A, the chromatogram of the original urine sample shows a large amount of hydrophilic material at the solvent front and two metabolites peaks, D1 and D2. As shown in Figure 10B, the chromatogram of the countercurrent extract with the coil planet centrifuge reveals enriched D1 and D2
262 COUNTERCURRENT CHROMATOGRAPHY / Solvent Extraction with a Helical Column See also: Countercurrent Chromatography: Overview. Original urine Fluorescence
Further Reading
D2
0
2
4
(A)
D1
6 8 10 Retention time (min)
14
D2 D1 dD3
Fluorescence
Countercurrent extract
12
demethyl-dD3
4-O -Glucuronide
0
2
(B)
4 6 8 10 Retention time (min)
12
14
Figure 10 Extraction of urinary metabolites of daunorubicin using the coil planet centrifuge. (A) HPLC analysis of the original urine; (B) HPLC analysis of the countercurrent extract.
peaks and three additional metabolite peaks, dD3, demethyl-dD3, and 4-O-glucuronide as indicated in the chromatogram.
Ito Y (1981) New continuous extraction method with a coil planet centrifuge. Journal of Chromatography 207: 161–169. Ito Y and Bhatnagar R (1981) Preparative countercurrent chromatography with a rotating coil assembly. Journal of Chromatography 207: 171–180. Nakazawa H, Riggs CE Jr., Egorin MJ, et al. (1984) Extraction of urinary anthracycline antitumor antibiotics with the coil planet centrifuge. Journal of Chromatography 307: 323–333. Ito Y (1988) Studies on hydrodynamic distribution of two immiscible solvent phases in rotating coils. Journal of Liquid Chromatography 11: 1–19. Ito Y and Conway WD (eds.) (1996) High-Speed Countercurrent Chromatography. New York: Wiley Interscience. Menet JM and Thibaut D (eds.) (1999) Countercurrent Chromatography. New York: Dekker. Du Q-Z, Wu P-D, and Ito Y (2000) Low-speed rotary countercurrent chromatography using a convoluted multilayer helical tube for industrial separation. Analytical Chemistry 72: 3363–3365. Berthod (ed.) (2002) Countercurrent Chromatography: The Support-Free Liquid Stationary Phase. Amsterdam: Elsevier. Du Q-Z and Ito Y (2003) Slow rotary countercurrent chromatography. Journal of Liquid Chromatography and Related Technologies 26(11): 1827–1838. Du Q-Z, Winterhalter P, and Ito Y (2003) Large convoluted tubing for scale-up of slow rotary countercurrent chromatography. Journal of Liquid Chromatography and Related Technologies 26(12): 1991–2002. Ito Y (2003) Spiral Disk for Dual Countercurrent Extraction. US Patent pending.
CRMs See QUALITY ASSURANCE: Reference Materials; Production of Reference Materials
CSV See VOLTAMMETRY: Cathodic Stripping
CYCLIC VOLTAMMETRY See VOLTAMMETRY: Linear Sweep and Cyclic