Anal. Chem. 1996, 68, 1207-1211
Affinity Countercurrent Chromatography Using a Ligand in the Stationary Phase Ying Ma and Yoichiro Ito*
Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 7N322, 10 Center Drive MSC 1676, Bethesda, Maryland 20892-1676
In countercurrent chromatography (CCC), an addition of a ligand to the liquid stationary phase remarkably improved both retention time and peak resolution of the analytes: various amino acid derivatives were separated by N-dodecanoyl-L-proline-3,5-dimethylanilide, while polar catecholamines and dipeptides were separated by bis(2-ethylhexyl)phosphoric acid. By selecting an appropriate ligand and dissolving it in the liquid stationary phase, the present CCC technique can perform a variety of separations comparable to chiral chromatography, ion chromatography, and affinity chromatography. Leakage of the ligand from the column can be entirely eliminated by introducing a small volume of a ligand-free stationary phase at the end of the column as an absorbent. The method further facilitates application of pH-zone-refining CCC and can increase the sample loading capacity over 10 times for a given column. Countercurrent chromatography (CCC) employs two immiscible solvent phases to separate analytes according to their partition coefficients (K).1-3 Success in separation depends on choice of the solvent system: first, the solvent system should provide a partition coefficient between 0.5 and 1, and second, the analytes should have significantly different K values. Usually, optimization of the partition coefficient is achieved by changing two parameters in the solvent system: relative hydrophobicity and either pH or ionic strength for charged analytes. A systematic search for an ideal solvent system by successively changing the hydrophobicity of the solvent system has been reported.3,4 This strategy, however, does not always lead to a suitable solvent system for an analyte mixture. The present paper introduces the use of a third parameter, affinity for a complexing agent or ligand, which selectively changes the partition coefficient of a particular group of compounds. Affinity separations have been used for many years in liquid chromatography. For example, affinity columns with a nucleotidebonded solid support are used for the separation of various nucleic acids and proteins.5 Chiral columns with chiral-selector-bound (1) Ito, Y. In Countercurrent Chromatography: Theory and Practice; Mandava, N. B., Ito, Y., Eds.; Marcel Dekker: New York, 1988; Chapter 3, p 79. (2) Conway, W. D. Countercurrent Chromatography: Apparatus, Theory, and Applications: VCH: New York, 1990. (3) Ito, Y. In Chromatography: Fundamentals and Applications of Chromatography and Related Differential Migration Methods; Heftmann, E., Ed.; Journal of Chromatography Library 51A; Elsevier: Amsterdam, the Netherlands, 1992; Chapter 2, pp A69-A107. (4) Oka, F.; Oka, H.; Ito, Y. J. Chromatogr. 1991, 538, 99-108. (5) Schott, H. Affinity Chromatography; Chromatographic Science Series 27; Marcel Dekker: New York, 1984. This article not subject to U.S. Copyright. Published 1996 Am. Chem. Soc.
solid phases are also used for the separation of enantiomers.6 In these techniques, the affinity ligand must be immobilized onto the solid stationary phase. Because this bonding process often requires several steps, the stationary phases are generally very expensive, and the method is usually limited to analytical-scale separations. We show here that the CCC technique is a versatile and cost-effective alternative, since it uses no solid support: the ligand can be held in place simply by dissolving it in the liquid stationary phase. As another example, if the analytes are charged compounds, a counterionic ligand may be used in analogy with ion chromatography. For separation of enantiomers, a chiral selector is dissolved in the stationary phase, comparable to the chiral stationary phase in chiral chromatography. For the separation of macromolecules such as proteins and nucleic acids, an affinity ligand such as a poly(ethylene glycol) derivative is introduced in the stationary phase to provide a system of affinity CCC with an aqueous-aqueous polymer phase system. All these CCC techniques have one important requirement: the ligand must be permanently retained in the stationary phase to avoid contaminating the eluate. This can be achieved by a suitable combination of ligand and two-phase solvent system. Some use of ligands in CCC has already been reported: separation of rare earth elements and heavy metals with bis(2-ethylhexyl)phosphoric acid7 and, more recently, chiral separations of amino acid derivatives with N-dodecanoyl-L-proline-3,5-dimethylanilide.8 In the present studies, these ligands are used in high-speed CCC to separate various biochemical compounds such as amino acid derivatives, peptides, and polar catecholamines. EXPERIMENTAL SECTION Reagents. Organic solvents, including methyl tert-butyl ether, n-hexane, ethyl acetate, methanol, and acetonitrile, were glassdistilled HPLC-grade and purchased from Burdick & Jackson Labs (Muskegon, MI). Hydrochloric acid, ammonium acetate, potassium phosphates, and bis(2-ethylhexyl)phosphoric acid (DEHPA) were analytical-grade from Sigma Chemical Co., (St. Louis, MO). Among analytes, dinitrobenzoyl(DNB)leucine and -phenylglycine, dinitrophenyl(DNP) amino acids, catecholamines, and dipeptides were also analytical-grade obtained from Aldrich (Milwaukee, WI), while DNB-valine, DNB-phenylalanine, and N-dodecanoyl-L-proline-3,5-dimethylanilide (DPA) were synthesized according to the method described by Oliveros et al.9 (6) Lindner, W. In Chiral Separations: Functional Aspects and Applications (Special volume); J. Chromatogr. A 1994, 666, 3-53. (7) Kitazume, E.; Bhatnagar, M.; Ito, Y. J. Chromatogr. 1991, 538, 133-140. (8) Ma, Y.; Ito, Y.; Foucault, A. J. Chromatogr. A 1995, 704, 75-81. (9) Oliveros, L.; Franco Puertolas, P.; Minguillon, C.; Camacho-Frias, E.; Foucault, A.; Le Goffic, F. J. Liq. Chromatogr. 1994, 17, 2301.
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Figure 1. Elution system for affinity CCC using a commercial high-speed CCC centrifuge.
Apparatus. Two different types of high-speed CCC centrifuges were employed: a commercial semipreparative model of the coil planet centrifuge (Ito multilayer separator/extractor, P.C. Inc., Potomac, MD) and a prototype of the semianalytical coil planet centrifuge equipped with a set of three column holders. The latter was fabricated at the NIH machine shop, but a comparable apparatus is available from Pharma-Tech Research Corp. (Baltimore, MD). Both instruments have a common design feature in that the column holder undergoes synchronous planetary motion, one rotation about its own axis during one revolution around the centrifuge axis. This particular mode of planetary motion prevents the flow tubes from winding, thus permitting continuous elution through the rotating column without the use of conventional rotary seals. The basic design of these instruments has been described in detail elsewhere.10 The commercial semipreparative unit (HSCCC, Figure 1) holds a single column holder (a) at 10 cm from the central axis of the centrifuge. A counterweight (b) consisting of a metal block and additional disks are placed on the opposite side of the rotary frame to balance the column weight. The column was prepared in our laboratory by winding 160 m long, 1.6 mm i.d. Tefzel tubing (Zeus Industrial Products, Orangeburg, SC) onto the holder hub, forming 16 coiled layers between a pair of flanges spaced 5 cm apart. The total capacity of the column measures 325 mL. In the present studies, the column was rotated at 800 rpm (∼70g at the column holder axis). The semianalytical high-speed CCC centrifuge is equipped with a set of three column holders, evenly arranged on the rotary frame at a distance of 7.6 cm from the centrifuge axis. This design provides an advantage over the above commercial model in that the columns are automatically balanced by themselves, hence no counterweight is required. Each multilayer coil consists of 11 layers of coils of 0.85 mm i.d. poly(tetrafluoroethylene) (PTFE) tubing (Zeus Industrial Products). Three columns are connected in series on the rotary frame to provide a total capacity of 180 mL. The column revolution of 1000 rpm (∼84g at the column holder axis) was applied throughout the present studies. Preparation of Solvent Systems and Sample Solutions. Each solvent system was prepared by equilibrating the solvent mixture, but excluding the ligand, in a separatory funnel at room temperature. After two clear layers were formed, the two phases were separated, and a desired amount of the ligand was added to the organic phase, which was then used as the stationary phase. The aqueous phase was used as the mobile phase without additives. The compositions of the two-phase solvent systems (10) Ito, Y. CRC Crit. Rev. Anal. Chem. 1986, 17, 65-143.
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Table 1. Two-Phase Solvent Systems, Ligands, and Partition Coefficients (K) of Analytes solvent systems
ligands
hexane ethyl acetate methanol 10 mM HCl
8 2 5 5
DPAa
MBEd 0.1 M NH4OAc,e 0.05 M HCl
1
DEHPA,f 1.5% epinephrine
(1.6 g/80 mL)
1 1
(-)-DNBb-Val (+)-DNB-Val (-)-DNB-phenyl-Gly (+)-DNB-phenyl-Gly (-)-DNB-Phe (+)-DNB-Phe (-)-DNB-Leu (+)-DNB-Leu DNPc-L-Orn DNP-DL-Glu DNP-L-Ala DNP-L-Pro DNP-L-Trp
1
MBE 0.1 M KH2PO4
analytes
DEHPA, 10%
K 0.35 0.87 0.41 0.87 0.53 1.57 0.66 2.87 0.06 0.11 0.30 0.45 0.63
DOPA 0.4
0.01
normetanephrine dopamine tyramine
0.5 0.8 1.7
Gly-Tyr Tyr-Gly Ala-Tyr Tyr-Ala Val-Tyr Tyr-Val Leu-Tyr Tyr-Leu
0.1 0.2 0.5 0.8 2.8 4.9 10 18
a N-Dodecanoyl-L-proline-3,5-dimethylanilide. b 3,5-Dinitrobenzoyl. c 2,4Dinitrophenyl. d Methyl tert-butyl ether. e Ammonium acetate f Bis(2ethylhexyl)phosphoric acid.
used in the present studies are listed in Table 1, together with partition coefficients of the samples applied. The sample solutions were prepared by dissolving the sample mixture (1-10 mg of each component) in 1-2 mL of the solvent, consisting of about equal volumes of each phase used for separation. Partition Coefficient Determination. Partition coefficients (K) were measured using the standard test tube procedure as follows: The two-phase solvent system was prepared as described above. A 2 mL aliquot of each phase, for a total of 4 mL, was delivered into a test tube (13 mm i.d. × 100 mm length), where a desired amount of the ligand and 1-2 mg of sample were added. The test tube was then stoppered, and the contents were mixed until the dissolved sample was completely equilibrated between the two phases. Using a vortex mixer, this required 10 successive applications at a few second intervals. The tube was then allowed to stand until two clear layers were formed. If necessary, the tubes were centrifuged for 2-3 min at 1000g to accelerate separation. An aliquot of each phase, usually 100 µL, was separately diluted
with 2 mL of a suitable diluent, usually methanol, and the absorbance was determined with a spectrophotometer (PM6 spectrophotometer, Zeiss, Hanover, MD). The partition coefficients were also estimated from the chromatogram using the following equation:
K ) (VR - Vm)/(VC - Vm)
(1)
where VR, Vm, and VC indicate the retention volume of the analyte, the volume of the mobile phase in the column, and the total column capacity, respectively. CCC Procedure. Figure 1 shows the elution system using a commercial model of the high-speed CCC centrifuge. CCC separations were performed as follows: The column was first entirely filled with the organic stationary phase using two different methods. In the first method, the column was entirely filled with the organic phase containing the ligand. This simple method results in continuous elution of the ligand in the effluent, even though it may not affect the detector. This complication is entirely eliminated in the second method, where a given amount of ligandfree stationary phase was first pumped into the column, followed by a known volume of the ligand-containing stationary phase (200 mL for the 1.6 mm i.d. column and 80 mL for the 0.85 mm i.d. column) by discharging an excess amount of the ligand-free stationary phase from the outlet of the column. Finally, the column contains a desired volume of the ligand-free stationary phase at the end of the column. During elution, a portion of this ligand-free stationary phase remains at the end of the column and absorbs the ligand from the flowing mobile phase to prevent its contamination of the eluate. After the column was filled with stationary phase, the mobile phase was pumped into the head of the column at a suitable flow rate (3.3 mL/min for 1.6 mm i.d. column and 1 mL/min for 0.85 mm i.d. column) while the column was rotated at the desired rate. After the solvent front emerged from the column, this blank run was continued until the polar impurities in the ligand were eluted and the uv trace returned to the base line. The sample solution was then injected through the sample port. If the amount of polar impurities in the ligand is negligible, then this preequilibrium procedure is not required, and sample injection may be made immediately after filling the column with stationary phase. The effluent was continuously monitored through a UV monitor (Uvicord S, LKB Instruments, Bromma, Stockholm, Sweden) at 254 or 280 nm and collected into test tubes at 2 min intervals using a fraction collector (Ultrorac, LKB Instruments). The separations can be repeated, if necessary, by injecting the sample in succession without renewing the stationary phase, as is routinely practiced in high-performance liquid chromatography. The control experiment for each separation was performed using a ligand-free stationary phase but under otherwise identical experimental conditions. RESULTS AND DISCUSSION Separation of Amino Acid Derivatives. Figure 2 shows chromatograms of four different DNB-amino acid racemates obtained with and without ligand in the stationary phase. The separations were performed with a solvent system composed of hexane/ethyl acetate/methanol/10 mM HCl (8:2:5:5 v/v) using the semianalytical high-speed CCC unit. With a ligand-free solvent system (Figure 2A), all components were eluted together close
Figure 2. Separation of four DNB-amino acid racemates by affinity CCC: (A) without ligand and (B) with ligand, DPA, in the stationary phase. Experimental conditions: apparatus, semianalytical highspeed CCC centrifuge equipped with three column holders at 7.6 cm orbital radius; columns, three multilayer coils consisting of 0.85 mm i.d. PTFE tubing connected in series with a total capacity of 180 mL; solvent system, hexane/ethyl acetate/ methanol/10 mM HCl (8:2:5: 5) without ligand (A) and with DPA (1.6 g) added to 80 mL of organic stationary phase (B); sample, 10 mg each of (()-DNB-amino acids indicated in chromatogram B; flow rate, 1 mL/min; revolution, 1000 rpm; detection, 280 nm.
to the solvent front, forming two partially resolved peaks. When a chiral selector, DPA, was added to the stationary phase (1.6 g/80 mL) (Figure 2B), all components increased in retention time and were well separated from impurities eluting near the solvent front. The four (-)-enantiomer peaks were well resolved from the respective (+)-enantiomer peaks, which were retained longer in the column. However, (+)-DNB-valine and (+)-DNB-phenylglycine were eluted together as a single peak due to their similar partition coefficient values in the present solvent system. The separation was completed in slightly over 7 h. Figure 3 shows similar chromatograms of five DNP-amino acids obtained from the same solvent system by the same semianalytical high-speed CCC unit. The left chromatogram (Figure 3A) was obtained from a ligand-free solvent system. The five components were eluted in three peaks, where the resolution between the second and third peaks was incomplete. Addition of a ligand (DPA) at 1.6 g/80 mL to the stationary phase substantially improved the peak resolution, as shown in the right chromatogram (Figure 3B). The first peak was partially resolved into two peaks, while the second and third peaks were completely separated into three peaks. The separation time was 2.5 h. In both separations, the ligand DPA introduced in the stationary phase modified the partition behavior of the analytes through hydrogen-bonding and/or π-electron interaction, resulting in increased retention time and higher peak resolution. Separation of Catecholamines. A group of polar catecholamines such as epinephrine partitions almost unilaterally into the aqueous phase, even in the hydrophilic 1-butanol/water two-phase solvent systems. Consequently, suitable partition coefficients are obtained only by saturating the aqueous phase with a salt such as barium chloride.11 The use of a ligand such as DEHPA Analytical Chemistry, Vol. 68, No. 7, April 1, 1996
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Figure 3. Separation of five DNP-amino acids by affinity CCC (A) without ligand and (B) with ligand (1.6 g) in the organic stationary phase. Sample: 2 mg each of five DNP-amino acids indicated in each chromatogram. Other experimental conditions are as in Figure 1. Figure 5. Separation of four isomeric pairs of dipeptides with (B) and without (A) ligand DEHPA. Experimental conditions: samples, 5 mg each of eight dipeptides indicated in the chromatogram; solvent system, methyl tert-butyl ether/0.1 M KH2PO4 (1:1), DEHPA (10%) added to the organic stationary phase; elution, HCl at 0.035 M added stepwise to the mobile phase after l h and at 0.1 M after 2 h. Other experimental conditions are as in Figure 4.
Figure 4. Separation of four catecholamines and one related compound by affinity CCC: (A) without ligand and (B) with 1.5% DEHPA in the organic stationary phase. Experimental conditions: apparatus, commercial semipreparative high-speed CCC centrifuge with 10 cm revolution radius; column, multilayer coil of 1.6 mm i.d. Tefzel tubing with a total capacity of 325 mL; solvent system, methyl tert-butyl ether/water containing ammonium acetate (0.1 M) and HCl (0.05 M) (1:1) (A) and 1.5% DEHPA added to the organic phase (B); sample, 5 mg each of five catecholamines indicated in chromatogram B (also see Table 1); flow rate, 3.3 mL/min; revolution, 800 rpm; detection, 280 nm.
alleviates this problem, as indicated in Table 1. Figure 4 shows chromatograms of four catecholamines and one related compound obtained by the semipreparative high-speed CCC unit. The separation was performed with a two-phase solvent system composed of methyl tert-butyl ether and an equivalent volume of (11) Weisz, A.; Markey, S. P.; Ito, Y. J. Chromatogr. 1987, 384, 132-136.
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the aqueous phase containing both ammonium acetate (0.1 M) and HCl (0.05 M). Without the ligand (Figure 4A), all components eluted at the solvent front as a single peak, as expected from their low partition coefficients. With DEHPA (1.5% v/v) in the stationary phase (Figure 4B), however, all components were completely resolved according to their K values shown in Table 1. Although these catecholamines are unstable in a basic solution, they are eluted as stable HCl salts with an acidic mobile phase. In this separation, the ligand DEHPA forms an ion pair complex with the catecholamine in the stationary phase, thus increasing the retention time of the analytes. This ionic interaction is comparable to ion chromatography, and the analytes are eluted in the increasing order of pKa and hydrophobicity. Separation of Isomeric Dipeptides. Four pairs of isomeric dipeptides were selected, all containing a tyrosine moiety to facilitate UV monitoring at 280 nm. These polar peptides are usually separated by CCC using 1-butanol/water systems with high viscosity and low interfacial tension, which results in low retention of the stationary phase. When these peptides were partitioned in a ligand-free methyl tert-butyl ether/phosphate buffer system, they were almost entirely distributed in the lower aqueous phase. However, introduction of ligand to the system remarkably increased their partition coefficients, as indicated in Table 1. Figure 5 shows the chromatogram obtained from a solvent system composed of methyl tert-butyl ether/0.1 M potassium phosphate buffer (1:1). To accelerate the elution of hydrophobic groups, dilute HCl was added to the mobile phase, first at a concentration of 0.035 M after 1 h and then at 0.1 M another hour later. Because of low partition coefficients, glycine derivatives were only slightly resolved near the solvent front. The rest of the peptides show higher peak resolution between the isomers. The method may be useful for separation of synthetic peptides.
In these separations, the use of a ligand further facilitates the application of pH-zone-refining CCC and increases the sampleloading capacity for these peptides over 10-fold for a given column.9,12,13 Consequently, the present method will be very useful in industrial applications for separation and purification of multigram quantities of products. Compared with analytical HPLC using a solid support, the present method requires considerably longer separation times, i.e., hours instead of minutes, but it provides important advantages over preparative HPLC, such as good sample recovery, high purity and high concentration of fractions, and cost-effective operation.
stationary phase, can be compared to various liquid chromatographic techniques, such as chiral chromatography, ion chromatography, and affinity chromatography. A major advantage of the CCC technique over these liquid chromatographic techniques is its simplicity, since there is no need to prepare an immobilized ligand on a solid support. One important requirement of the present method is careful selection of the two-phase solvent system which can effectively retain the ligand in the stationary phase. Minor leakage of the ligand to the mobile phase can be corrected by placing a small volume of ligand-free stationary phase at the end of the column as an absorbent.
CONCLUSIONS As described above, addition of a suitable ligand to the stationary phase results in marked improvement of retention and peak resolution of analytes which are not easily separable by the conventional high-speed CCC technique. The present CCC method, which allows a liberal choice of the ligand in the
ACKNOWLEDGMENT The authors thank Dr. Henry M. Fales for editing the manuscript.
(12) Ma, Y.; Ito, Y. J. Chromatogr. A, in press. (13) Ito, Y. In High-Speed Countercurrent Chromatography; Ito, Y., Conway, W. D., Eds.; Wiley Interscience: New York, 1995; Chapter 6, pp 121-175.
AC9509263
Received for review September 13, 1995. January 10, 1996.X
X
Accepted
Abstract published in Advance ACS Abstracts, February 15, 1996.
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