Journal of Chromatography. 260 (1983) 419—42?
`Eisevier Science Publishers B.V., Amsterdam — Printed in the Netherlands
`
`CHROM. H.668
`
`COMPARISON OF SEVERAL HIGH-PERFORMANCE LIQUID CHROMA-
`TOGRAPHY TECHNIQUES FOR THE SEPARATION OF OLIGODEOXY-
`NUCLEOTIDES ACCORDING TO THEIR CHAIN LENGTHS
`
`WOLFGANG HAUPT and ALFRED PINGOUD'
`
`Abteilung Biophysikm‘ische Chemie. Zenrrwn Biochemfe. Medizinische Hoehschuie Hammer. Konstamy-
`GurschowlSn-asse, 0-3000 Hmover 6! (G.F.R.)
`
`(Received December 10th, 1982)
`
`
`
`SUMMARY
`
`The use of reversed-phase, reversed-phase—ion-pair and anion-exchange high-
`performance liquid chromatography (HPLC) was investigated for the analytical and
`preparative separation of oligodeoxynucleotides according to their chain length. The
`data obtained with homooligonucleotides and oligonucleotides of defined sequence
`show that reversed-phase—ion-pair and anion—exchange, but not reversed-phase,
`HPLC can be used reliably to separate oligodeoxynucleotides according to their chain
`length, largely irrespective of their base composition. The chain length limits for
`complete separation within 30 min by reversed-phase, reversed-phase—ion-pair and
`anion-exchange HPLC are approximately 10, 15 and 20 nucleotides, respectively.
`
`
`INTRODUCTION
`
`High-performance liquid chromatography (HPLC), because of its high reso-
`lution, reproducibility and ease of operation, has become an indispensible method
`for the separation of oligoderynucleotides, for both analytical and preparative pur-
`poses. A variety of different techniques have been developed, based on reversed-phase
`HPLC ‘ ‘3, reversed-phase—ion-pair HPLC” , stronganion-exchange HPLC5‘9 and size
`exclusion HPLC”, and have been used so far mainly for the separation of short
`oligonucleotides. The separation of oiigodeoxynucleotides by these techniques is de-
`termined by the base composition and chain length of the oligodeoxynucleotides to
`be separated, which in turn determine their polarity and size. These properties, albeit
`to difi'erent extents in the various chromatographic systems, control the degree of
`retention of a particular oligodeoxynucleotide. The retention mechanisms involved
`are not fully understood.
`Reversed-phase HPLC of oligodeoxynucleotides is dominated by hydrophobic
`interactions between the solute and the bonded phase‘; it is more sensitive to the
`base composition than reversed-phase—ion-pair, anion-exchange and size exclusion
`HPLC. Nevertheless the retentic-n sequence of oligonucleotides with similar base
`composition is controlled by the charge of the oligonucleotide.
`In reversed-phase—ion-pair HPLC, negative charges on the oligodeoxynucleo-
`
`002l—96'I3f83f303110
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`© 1933 Elsevter Seience Publishers B.V.
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`W. HAUPT, A. PINGOUD
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`tide are neutralized by positively charged alkylammonium ions. It is not clear, how-
`ever, whether this process occurs in the mobile phase1 1 or in the stationary phase”,
`leading to dynamic ion exchange. The retention is determined mainly by the charge
`of the oligodeoxynucleotide; in addition to these dominating electrostatic interac-
`tions, hydrophobic interactions between the oligodeoxynucleotides and the reversed
`phase also play a role in the chromatographic processs.
`The separation of oligodeoxynucleotides by anion-exchange HPLC is depen-
`dent on differences in charge; as currently used anion exchangers are based on a silica
`gel matrix, in which the tertiary ammonium moiety is connected to the resin via an
`aliphatic spacer, there are hydrophobic interactions in addition to the ionic interac-
`tions. The inclusion of non-polar solvents in the mobile phase diminishes the effect
`of the reversed phase on the chromatographle-l“.
`Recently, silica gel bonded polyol phases have become commercially available
`for the separation of oligodeoxynucleotides by size exclusion HPLC. The separation
`can be carried out with a variety of mobile phases. The resolution depends on the
`mobile phase, indicating that size is not the only determinant for the chromatogra-
`phy. For small and medium sized oligodeoxynucleotides the resolution is not as good
`as with the above-mentioned chromatographic systems”.
`One characteristic feature of a particular oligodeoxynucleotide, and one that
`can be used conveniently for the purpose of analysis or preparative purification, is
`its size. It is therefore desirable to have chromatographic systems that allow sepa-
`rations according to size, 118., chain length. A separation in such a chromatographic
`system ideally should be unaffected by base composition and should allow separation
`with high resolution over a wide range of chain lengths. For this purpose we inves-
`tigated the suitability of two standard and one new chromatographic system for chain
`length-dependent separations of oligodeoxynucleotides, namely (1) reversed-phase
`HPLC on Zorbax ODS columns, (2) reversed-phase—ion-pair HPLC on LiChrosorb
`RP-8 columns and (3) anion-exchange HPLC on Partisil SAX columns. We used
`commercial homooligodeoxynucleotides of specified chain lengths as well as chemi—
`cally or enzymatically synthesized oligodeoxynucleotides of defined sequence to study
`the limits of resolution of these chromatographic systems.
`The results show that both reversed-phase~ion-pair HPLC and anion-exchange
`HPLC, but not reversed-phase HPLC, can be used reliably to separate oligodeoxy-
`nucleotides according to their chain length, with only slight interference by base
`composition. Whereas, reversed—phase—ion—pair HPLC has a cut-off for an acceptable
`resolution at a chain length of around l5—20 nucleotides, anion-exchange HPLC
`allows even longer oligodeoxynucleotides to be separated.
`
`EXPERIMENTAL
`
`Instrumentation
`
`Two chromatographs were used: ( l) a DuPont 850 liquid chromatograph, con-
`sisting of a gradient controller, a three-head pump, a temperature-controlled column
`compartment, a UV spectrophotometer and a Rheodyne 7125 injection valve, and
`(2) a Pye Unicam liquid chromatograph, consisting of an LC-XP gradient program-
`mer, an Altex Model 100A pump, an LC—UV detector and a Rheodyne 7125 injection
`valve, to which a Merck LiChrocart-Autofix pneumatic mounting device was at-
`tached.
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`HPLC 0F OLlGODEOXYNUCLEOTlDES
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`Chemicals and oligonudeotides
`All homodeoxyoligonucleotides were obtained from PJ... Biochemicals (St.
`Goar, G.F.R.). d(GGAATTCC) and d(CGAATTCGi were kindly given by Dr. H.
`Bloecker and Dr. R. Frank (GBF, Stockheim, G.F.R.) and by Dr. M. Zabeau
`(EM BL, Heidelberg, G. F.R.), respectively. Tetrabutylammonium hydrogen sulphate
`(TBAHSO4),
`triethylamine and formamide were products of Fluka (Neu-Ulm,
`G.F.R.) and were used without further purification. HPLC-grade acetonitrile was a
`product of Burdick & Jackson Labs. (Muskegon, MI, U.S.A.) and was provided by
`Macherey, Nagel & Co. (Dijren, G.F.R.). T4 DNA ligase was obtained from BRL
`(Neu-lsenberg, G.F.R.). All other chemicals used were of pro analysi grade from
`Merck (Darmstadt, G.F.R.).
`
`Columns and chromatographic conditions
`All eluents were filtered through a Whatman GF}r F glass-microfibre-filter (Vet-
`ter, Wiesloch, G.F.R.) and degassed prior to chromatography.
`For reversed-phase separations Zorbax ODS columns (25 cm X 4.6 mm I.D., 5
`Jum) from DuPont (Bad Nauheim, G.F.R.) were used. The triethylammonium ace-
`tate (TEAA) buffer was prepared as described elsewhere‘. Solvent A was 0.1 M TEAA
`(pH T.0}--l% acetonitrile and solvent B was 0.1 M TEAA (pH 'i'.0)——50% acetonitrile.
`If not stated otherwise, a linear gradient from 15 to 30% B in 30 min was applied.
`The flow-rate was 1.0 mlfmin. Chromatography was carried out at ambient temper-
`ature.
`
`Reversed-phase—ion-pair separations were performed on Hibar LiChrocart
`cartridges (25 cm x 4 mm ID.) packed with LiChrosorb RP-S, 10 ,um (Merck). So]-
`vent A was 50 mM potassium phosphate (pl-I 5.9)—2 mM TBAHSO4 and solvent B
`was 50 mM potassium phosphate (pH 6.5)—2 mM TBAHSOa—600/n acetonitrile. The
`standard linear gradient used was from 15 to 80% B in 60 min. The flow-rate was
`1.0 ml/min. Chromatography was carried out at ambient temperature.
`Whatman Partisil 10 SAX columns (25 X 4.6 mm I.D., 10 um) obtained from
`IC-Chemikalien (Munich, G.F.R.) were used for anion-exchange HPLC. Solvent A
`was 1 mM potassium phosphate (pH 6.3) in formamide—water (6:4) and solvent B
`was 0.3 M potassium phosphate (pH 6.3) in formamide—water (6:4). The linear gra-
`dient used was from 0 to 80% B in 60 min. The flow-rate was 1.0 ml/min. Chro-
`matography was carried out at 45°C.
`
`RESULTS
`
`Figs. 1 and 2 show chromatograms of mixtures of oligo-(dA)s and oligo-(dT)s
`separated by reversed-phase chromatography. Oligonucleotides of different chain
`length are well resolved within the oligo-(dA) and oligo-(dT) series up to a chain
`length of at least ten nucleotides under the conditions given.
`Oligo-(dT)s are considerably more retarded than oligo-(dA)s of the same chain
`length. Thus, for example, p(dA)m is eluted before p(dT)(,. This demonstrates that
`the base composition of the oligonucleotide has a considerable effect on the retention
`of the oligonucleotides. This elTect, of course, can be of advantage when oligomers
`of similar chain length or even sequence isomers have to be separated. This is illus-
`trated in Fig. 3, where a separation of d(GGAATTCC) from its sequence isomer
`
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`O
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`10
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`min
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`0
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`10
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`20
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`30
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`min
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`Fig. l. Reversed—phase HPLC of oligo-(dA)s. Column: Zorbax ODS (25 cm X 3.6 mm I.D.). Eluents: A,
`0.1 M TEAA (pH) TKO—1% acetonitrile; B, 0.1 M TEAA (pH 7.0}50'14: acetonitrile; linear gradient from
`15 to 30% B in 30 min at ambient temperature; flow-rate, 1.0 mllmin. Unidentified peaks are due to
`contaminants in the individual samples.
`
`Fig. 2. Reversed-phase HPLC of oligo-(dns. Chromatographic conditions as in Fig. l.
`
`d(CGAATTCG) is shown. In fact, our analysis of commercially available oligo-(dT)s
`and in particular of oligo-(dA)s has shown that these preparations contain consider-
`able amounts of impurities (cf, Figs. 1 and 2), which are not detected by polyacryl-
`amide gel electrophoresis under denaturing conditions. by homochromatography
`after labelling the 5’-end with [“P]phosphate or by anion-exchange HPLC (see be-
`low), suggesting that they consist of chemical derivatives of these oligonucleotides
`with the same chain length or charge.
`Figs. 4 and 5 show separations of mixtures of oligo-(dA)s and oligo-(st in
`a reversed-phase—ion-pair system. The individual oligonucleotides are extremely well
`resolved up to a chain length of at least 16 nucleotides, the peaks being sharper and
`more symmetrical than in the reversed-phase system (compare Figs. 4 and l or Figs.
`5 and 2). Oligo—(dA)s are slightly more retained than oligo-(dT)s, e.g., p(dA)g is
`eluted after p(dT)10. The influence of base composition is less pronounced in the
`reversed-phase system. Accordingly, reversed-phase—ion-pair HPLC is not as useful
`as reversed-phase HPLC for the separation of oligonucleotides of different base com-
`position but similar chain length. This is also apparent from Fig. 6, which shows the
`
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`HPLC OF OLIGODEOXYNUCLEOTIDES
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`20
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`min
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`0
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`30
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`min
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`Fig. 3. Reversed-phase HPLC of the sequence isomers d(GGAA‘ITCC) and d(CGAATTCG). Column:
`Zorbax ODS (25 cm X 4.6 mm I.D.). Eluents: A, 0.1 M TEEA (pH 7.0}!% aeetonitrile: B, 0.I M TEAA
`(pH 7.0}50‘3’3 acetonitrile; linear gradient from 20 to 25% B in 20 min at ambient temperature; flow-rate.
`l mlfmin.
`
`Fig. 4. Reversed~phase ion-pair HPLC of oligo-(dAk. Column: LiChrocart cartridge (25 cm X 4.6 mm
`ID.) filled with LiChrosorb RP-S. Eluents: A. 50 mM potassium phosphate (pH S.9}—2 mM TBAHSO‘;
`B. 50 mM potassium phosphate (pH 6.5}2 mM TBAHSO4—60‘Vn aoetonitrile; linear gradient from l5 to
`30% B in 60 min at ambient temperature; flow-rate, 1.0 rnlfrnin.
`
`reversed-phase—ion-pair chromatogram of a mixture of d(GGAATTCC) and
`d(CGAATTCG). Although the retention time is longer than in the reversed-phase
`chromatography shown in Fig. 3, the separation is not as good.
`Figs. 7 and 8 show chromatograms of mixtures of oligo-(dA)s and oligo-(dT)s
`separated by strong anion-exchange HPLC. The retention times under the chosen
`conditions are nearly constant with respect to base composition, e.g., p(dA)m is
`eluted at the same position as p(dT)10. The resolution of oligonucleotides of increas-
`ing chain length is excellent up to a chain length of about 20. The high resolution
`is also demonstrated in Fig. 9, which shows the chromatogram of the separation of
`commercial oligo—(dA).. More than 30 discrete peaks can be seen. The usefulness of
`ion-exchange HPLC for the separation of longer oligonucleotides is particularly
`apparent from Fig. 10. In the HPLC run shown the mixture of products resulting
`from the T4 DNA ligase catalysed oligomerization of pd(GGAATTCC) was separ-
`
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`dtfifihATTCC]
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`0.02
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`a E i
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`= e
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`D
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`min
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`0
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`30
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`Min
`
`Fig. 5. Reversed-phase—ion-pair HPLC of oligo-(dns. Chromatographic conditions as in Fig. 4.
`
`Fig. 6. Reversed-phase—ion-pair HPLC of the sequence isomers d(GGAATTCC) and d(CGAATTCG).
`Chromatographic conditions as in Fig. 4.
`
`ated by anion-exchange HPLC. Resolution was achieved up to the “octamer”, 5.9.,
`up to a chain length of 64 nucleotides.
`We have also used commercial preparations of oligodeoxynucleotides of the
`(dG) and (dC) series in reversed-phase, reversed-phase—ion-pair and anion-exchange
`HPLC. In general, the oligonucleotides of defined chain length as stated on the label
`proved to be much less homogeneous than the corresponding (dA)- and (dT)—oli-
`godcoxynucleotides. Nevertheless, similar results were also obtained with the oligo-
`nucleotides of the (dG) and (dC) series compared with those of the (dA) and (dT)
`series (data not shown).
`
`DISCUSSION
`
`We have compared the utility of three different chromatographic systems
`for the separation of oligodeoxynucleotides by HPLC according to chain length. It
`was not our aim to improve previously developed chromatographic system, but rath-
`er to use them, with minor modifications, in order to establish their limits of reso-
`lution with respect to size and base composition. Our results show that within a series
`of homooligonucleotides all three systems reliably lead to separation according to
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`HPLC 0F OLIGODEOXYNUCLEOTIDES
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`20
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`0
`min
`30
`20
`10
`0
`Fig. 7. Anion-exchange HPLC of oligo-(dAk. Column: Whatman l SAX (25 cm x 4.6 mm ID). Eluents:
`A, 1 mM potassium phosphate (Ph 6.3) in formamide—water (6:4); B, .3 M potassium phosphate (Ph 6.3)
`in formamidewater (6:4); linear gradient from to 8% in 6 min at 45°C; flow-rate. l. nil/min.
`
`min
`
`Fig”. 8. Anion-exchange HPLC of oligo-{dT}s. Chromatographic conditions as in Fig. 7.
`
`chain length. The chain length limits for separation by reversed-phase, reversed-
`phase—ion-pair and strong anion-exchange HPLC are ca. 10, 15 and 20 nucleotides,
`respectively. These limitis apply for the complete separation of homologous oligo-
`deoxynucleotides within ca. 30 min. With suitable modifications, e.g., by using dif-
`ferent gradients, these limits may well be extended towards longer chain lengthslt
`is known from the separation of short oligonucleotides that the retention of oligo-
`deoxynucleotidtm in reversed-phase HPLC is very sensitive to the base composition‘.
`Our results show that oligo-(dT)s are much more strongly retained on Zorbax ODS
`(this work) and LiChrosorb RP-S columns (data not shown) than oligo-(dA)s. The
`retention of oligodeoxynucleotides in reversed-phaseiion-pair HPLC, on the other
`hand, is only slightly influenced by the base composition. Interestingly, our results
`indicate that with this chromatographic system oiigo-(dA)s are more strongly re-
`tained than oligo-(dT)s, probably a consequence of the diflerent base stacking”,
`which might affect the accessibility of the phosphate residues of the oligodeoxynu-
`cleotides for the ion-pairing agent.
`Under the conditions applied the retention of oligodeoxynucleotides is least
`affected by the base composition of the sample in anion-exchange HPLC. It has been
`
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`426
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`W. HAUPT. A. PINGOUD
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` 0
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`20
`
`to
`
`min
`
`Fig. 9. Aniomexchange HPLC of a material that was designated by the supplier as p(dA)25- 3o. Column
`and eluent as in Fig. 7*; linear gradient from 0 to 100% B in 75 min at 45°C; flow-rate, l mlfmin.
`
`0.03
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`0.01
`
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`D
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`min
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`ll]. Anion-exchange HPLC of the product mixture of the T4 DNA ligase catalysed oligomerization
`Fig.
`of pd(GGAATTCC) that was obtained by incubating 6 FM [”P]pd(GGAA'ITCC) and 2.4 ,uM
`d(GGAATTCC) at 22°C for l6 h with 48 unitsfml of T4 DNA ligase in a mixture of40 mM Tris HC] (pH
`7.6), 8 mM magnesium chloride, 1 not! dithioerythritole (DTE) and 470 _ul'n ATP. The separated peaks
`were identified by polyacrylamide gel electrophoresis“: a = d(GGAATTCC) monomer; b = dimer; c
`= trimer; d = tetramer; c = pentamer; f = hexamer; g = heptamer; a’ = AMP-pd(GGAATTCC)
`ligation intermediate. Analysis of the fractions by Cerenkov radiation in a scintillation counter indicated
`that oligonucleolides beyond the octamer were resolved. The small satellite peaks of peaks :1. a'. b, c. d
`and e probably are due to slight heterogenein in the starting material.
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`HPLC OF OLIGODEOXYN UCLEOTIDES
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`42?
`
`reported, however, that under different conditions, in particular in the absence of
`organic solvents in the mobile phase, anion-exchange HPLC can by used to separate
`sequence isomers of small oligonucleotidess" 7. This demonstrates that the inclusion
`of organic solvents in the mobile phase is necessary to suppress hydrophobic inter-
`actions between the sample and the stationary phase‘““.
`Taken together, our results suggest that for short to medium sized oligonu-
`cleotides reversed-phase—ion-pair and anion~exchange HPLC are equally suitable for
`separations according to chain length (cf. also ref. 18), whereas for longer oiigonu-
`cleotides anion-exchange HPLC is superior to reversed-phase—ion-pair HPLC. It
`must be mentioned in this context, however, that anion-exchange HPLC columns
`seem to suffer from shorter Iifetimes“ than reversed-phase HPLC columns. Thus, for
`analytical or preparative work with small
`to medium sized oligonucleotides re»
`versed-phase and reversed-phase ion-pair HPLC is the combination of choice, one
`system separating according to polarity and size, the other predominantly according
`to size. For separations of larger oligonucleotides by HPLC there is as yet no alter-
`native to anion-exchange or size exclusion HPLC.
`
`ACKNOWLEDGEMENTS
`
`We thank Dr. W. Block for his active support during the initial phase of this
`work. The expert technical assistance of Mr. R. Mull is gratefully acknowledged.
`This work was supported by grants from the Deutsche Forschungsgemeinschaft and
`the Fonds der Chemischen Industrie.
`
`REFERENCES
`
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`G. D. McFarland and P, N. Borer, Nucl. Acid Res., 7 (1979} I067.
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`10 D. Molko, R. Derbyshire, A. Guy, A. Roget, R. Teoule and A. Boucherle, J. Chroma:ng 206 { l98l)
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`ll Cs. Horvtith, W. Melander, [. Molnar and P. Molnar, Anal. Chem, 49 (1979) 2295.
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`18 H. J. Fritz, D. Bid: and W. We”, in H. G. Gassen and A. Lang (Editors), C't‘mrrtt‘ct:ttf and Enzymatic
`Synthesis of Gene Fragments, Verlag Chemie, Weinheim, 1982, p. I99.
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