Slalom Chromatography: An Overview

IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY Available online at www.ijrpc.com Review Article SLALOM CHROMATOGRAPHY: AN OVERVIEW Vineeta Khanvilkar, Aditi Chitnis*, Abhay Shirode and Vilasrao Kadam Bharati Vidyapeeth’s College of Pharmacy, Sector 8, C.B.D., Belapur, Navi Mumbai, Maharashtra, India. ABSTRACT Various techniques like gel electrophoresis, gel permeation chromatography, ion-exchange chromatography have been employed for analysis and separation of large biomolecules such as double-stranded DNA and RNA. A technique called Slalom chromatography was discovered in 1988 by Boyes et al and Hirabayashi and Kasai independently. This size-fractionation method includes reptation of DNA fragments through the column packing which resembles a person on skis going down a slope and turning quickly around flags. The principle mode of separation is based on hydrodynamic phenomenon rather than an equilibrium one. The reorientation time of DNA determines the orders of elution i.e. larger strands are eluted after the smaller ones. The article reviews about the chromatographic conditions used for slalom chromatography and various factors affecting the DNA separation. The physicochemical factors have been shown to have a critical effect on the separation DNA topology, temperature, mobile phase viscosity and particle size of packing material. The separation technique reviewed provides a new effective tool for physicochemical and hydrodynamic studies of DNA. Keywords: Slalom chromatography, size-fractionation, DNA, hydrodynamic phenomenon. INTRODUCTION eventually result in size-dependent separation. Biochemical analysis techniques refer to a set of Ion-exchange chromatography is categorized methods, assays, and procedures that enable under first group whereas size exclusion and scientists to analyze the substances found in slalom chromatography are truly size-dependent living organisms and the chemical reactions modes. Chromatography is versatile as a underlying life processes. To perform a research tool for both preparation and analysis comprehensive biochemical analysis of a because as soon as a separation process is biomolecule in a biological process or system, complete, valuable information is also obtained the biochemist typically needs to design a on aspects such as quantity, quality and strategy to detect that biomolecule, isolate it in composition. Therefore, the more sophisticated pure form from among thousands of molecules the chromatographic mode, the higher the that can be found in an extracts from a biological quality of the information obtained. sample, characterize it, and analyze its function. For the analysis and separation of large Size of the nucleic acids biological macromolecules such as DNA and The term "size" can have a variety of meanings. RNA, various techniques have been developed In biochemistry, it is generally used to mean over the past few years. Chromatographic either molecular mass or degree of modes so far used are divided into two groups: polymerization of macromolecules, The size of purely size-dependent modes and modes that polynucleotides has been exclusively expressed 66 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 by the degree of polymerization, e.g. number of been difficult. Most DNA fragments cannot base pairs or kilobasepairs (abbreviated as bp permeate the pores of the ion ex-changer and or kbp) in the case of double-stranded DNA. this resulted in too low an operational capacity. It Polynucleotides take different shapes depending remains inefficient because of poor resolution on their degree of polymerization. A small and slow speed. The amount of sample that can double-stranded DNA fragment, e.g. 20 bp (2 x be used is rather limited, because the capacity 6.4 nm), has a rigid and globular shape. Large of non-porous adsorbents is generally not so double-stranded DNA fragments are fibrous, and high hence low loading capacity5. it is becoming difficult to treat them as globular molecules. A fragment with ca. 100 bp (2 x 34 3) Size-exclusion (gel-permeation) nm) should be treated as a rod with a certain chromatography: It separates biomolecules on degree of elasticity. A long double-stranded the basis of true size difference. The column DNA molecule has a kink at every ca. 50 nm consists of a hollow tube tightly packed with (150 bp) and consequently forms a random coil, extremely small porous polymer beads designed 1,2 which is the most favourable shape .The to have pores of different sizes. Small random coil is very flexible, and its shape molecules of analyte can enter the pores of gel changes without a break between the contracted more easily and therefore spend more time in and slightly extended forms by an external force these pores increasing their retention time. provided by Brownian motion of water Conversely, large analytes spend little time in molecules. the pores and elute quickly. Disadvantages: The size of the nucleic acids of Chromatographic modes currently applicable interest is usually too large to be applicable to a for size-dependent separation of nucleic gel-permeation column. The resolution acids attainable by gel permeation chromatography is 1) Gel electrophoresis: Nucleic acids are usually inferior to that of other modes, such as separated by applying an electric field to move ion-exchange and reversed-phase.It is difficult to the negatively charged molecules through an speed up the operation because it is essential to agarose matrix. Shorter molecules move faster maintain an equilibrium state of distribution of and migrate farther than longer ones because the target molecules between two phases, i.e. shorter can migrate more easily through the stationary phase retained in intrapore spaces pores of gel. Disadvantages: It takes longer for and the mobile phase6. both separation and recovery, and the recovered The comparison of chromatographic modes sample is usually contaminated with impurities available for size fractionation of nucleic acids is contained in the Agarose. shown in table 1. 2) Ion-exchange chromatography: Separation SLALOM CHROMATOGRAPHY of nucleic acids by ion–exchange Slalom chromatography (SC) was discovered chromatography is mainly based on electrostatic independently by Boyes et al7 and Hirabayashi interactions between the phosphate groups of and Kasai in 1988. It is a chromatographic mode the former and the positively charged groups of for separation of flexible biological molecules. In the ion exchanger. Adsorbed oligonucleotides this technique, the reptation of the DNA were eluted by a salt gradient in the order of the fragments through the column packing follows degree of polymerization. Ion exchange HPLC the flow direction and its like a snake edging is separates oligonucleotides according to number way into long grass. This new mode of of charged groups (phosphate linkages)3. The separation was named "slalom separation decreases with oligonucleotide chromatography", because the proposed model length. Anion-exchange chromatography with a reminds us of a person on skis going down a 8 proper gradient is also widely used for slope and turning quickly around flags and can purification of RNA and DNA, with plasmid be illustrated as figure. 1. The separation of DNA4. molecules by liquid chromatography is classically based on an equilibrium phenomenon Disadvantages: The separation of double- (determined by the distribution coefficient) 9 stranded DNA fragments larger than several between the eluent and the stationary phase . In hundred base pairs by ion-exchange this method, the separation occurs via a chromatography on macroporous supports has hydrodynamic phenomenon rather than an 67 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 equilibrium one. Slalom chromatography can are unfolded and extended owing to the laminar separate in a shorter space of time larger flow generated by the solvent passing through double-stranded DNA molecules (ranging from the narrow channels. For example, a 10 kbp roughly 5 to 50 kbp) than the conventional fragment will become 3.4 µm at maximum. chromatographic procedures by using a column These extended molecules must flex quickly for gel permeation with an ordinary HPLC under a fast flow of mobile phase to pass system. The order of elution is opposite to that through the openings. If we use a 30 cm column expected for gel permeation chromatography; packed with particles of 10 µm diameter, DNA the larger DNA fragments are eluted later than molecules should turn as many as 36,000 times, 10 the smaller ones . Theoretical background of because the number of layers of particles reach SC is still giving its first steps and the transient 1200 per centimetre of column length. A condition between HDC and SC is under fragment that has a retention time of 10 min will investigation11. turn 60 times per second. It is quite possible that Important characteristics of slalom the longer the DNA molecule, the more difficulty chromatography revealed by extensive studies it encounters in passing through the openings. are summarized below: Therefore size-dependent separation in the 1) DNA fragments do not interact with the opposite order to gel chromatography should column packing. occur. However, the elution order in HDC is the 2) Only the particle size of the packing is same as in gel permeation chromatography due important. Smaller particles can resolve to the exclusion of the large polymer from the smaller DNA fragments and larger low velocity regions near the particle wall15. particles can resolve larger DNA fragments. MODEL 3) Pore size and the chemical nature of In this model, the column packing is treated as a packing particles are not important. three-dimensional network of pores with an 4) Separation depends largely on the flow- average diameter or length (l). This l diameter is rate: the higher the flow-rate, the more linked to the average particle diameter dp and the DNA fragments are retarded. The the interparticle porosity n by temperature also has an influence on the separation1. l = 0.42dp n / n-1 THEORY The DNA molecule is represented as in the The separation depends on the flow-rate and the reptation theory of de Gennes by a chain of p particle size of the column packing and not on segments each of contour length l. The their pore size or chemical nature. In this case, sequence of pores followed by the chain is the column packing is only used for the called the tube with a total length equal to pl. formation of the network of narrow interstices Reptation theory describes effect of polymer between spherical particles. When a DNA chain chain entanglements on the relationship is applied to a chromatographic system, it between molecular mass and chain relaxation frequently turns around the spherical obstacles; time. The theory envisions that the movement of the larger the fragments or the smaller the entangled polymer chains is analogous to particle size, the more difficult it is for the snakes slithering through one another16. As the fragments to travel across the interstitial spaces DNA fragment was aligned in the flow direction it created inside the column. According to this was assumed to be a linear stretching of the description, this separation mode cannot be DNA fragment in one pore. ∆li is the linear explained in terms of an equilibrium constant progression length of the DNA fragment in pore i between the mobile and the stationary phases. when the mobile phase velocity variation Another chromatographic technique i.e. column increases from vi to v (∆vi = v- vi). When the hydrodynamic chromatography (HDC), used for mobile phase velocity increases by dv, ∆li the separation of polymers or particles, shows increases by d ∆li and p increases by dp in the same characteristic. This technique is also accordance with SC mechanism. based on the use of a laminar flow which occurs The differential equations describing this in the interstitial spaces created between the phenomenon can be written as particles packed in the column12-14. When the DNA molecules are applied to the column, they dp / dv = k( p∞ - p) 68 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 reorientation hence cover a longer distance than 19 d∆li / d∆vi = k’ larger molecules . The larger is the DNA molecules, the longer the reorientation time. If J where k and k’ are positive constants and p∞ is is the curvilinear co-ordinate along the tube, the the value corresponding to the maximum DNA reduced curvilinear co-ordinate j is defined as stretching. To model the DNA retention inside the column, a j = J/Pr parameter ‘Relative retention Time (RRT)’ was introduced and by the definition, When j varies from 0 to 1, the fraction of j oriented along x is replaced by the same fraction RRT = tR / tR,0 or along x. On average the tube is oriented along the diagonal z as reported by Viovy et al20. The RRT = tR / tNR DNA segment that leaves the tube aligns itself preferentially in the field direction to minimize its Where tR is the retention time of a particular potential energy. DNA molecule and tR,0 or tNR The flow rate dependence of macromolecules of is that corresponding to flowthrough fraction different size can be explained in terms of (non-retained molecules). deformation and orientation of simple The resolution of a pair of DNA fragments can macromolecular models in steady uniaxial be measured from the chromatogram using the elongation. The strength of the flow can often be retention time tR and peak width ω by the correlated with Deborah number, De: a ratio of following equation, the hydrodynamic forces to the Brownian forces. Deborah number is defined as ratio of relaxation RS = 2( tRB – tRA ) / (ωA + ωB ) time characterizing the time it takes for a material to adjust to applied stresses or Where the subscripts A and B refer to the first deformations21. Significant molecular stretching and the second eluting peaks, respectively. in steady flows occurs only when De > 0.5. A Assuming that the two peaks have a similar relaxation effect in the macromolecular flow is bandwidth, i.e. ω =ωA + ωB , it can also be characterized by the Deborah number that can assumed to be related to the column plate be presented as the ratio of a moving object number N determined on the B peak by the well relaxation time θ to the time when the object known equation17, was exposed to deformation θp N = 16 (tRB2/ωB2) De = θ/θp The progression of a fragment through the For macromolecules θ is directly proportional to closed column packing can be modeled as the boundary viscosity at zero shear velocity and shown in figure 2. There are two cases: 1) when to the molecular weight and is inversely the chain is short, it is aligned in the direction x1 proportional to the molecular concentration22 of the flow before it is switched from an average whereas θp is defined by porous media value of angle θ to the new direction of the flow properties: x2 , then the fragment progression is considered as a sequence of independent oriented paths θp = a. ε. dp/u with no or only weak retardation. 2) When the chain is sufficiently large, it is partially aligned Where, ε is the bed porosity, dp is the particle along x1 before the flow turns around the diameter and a is a numerical coefficient. particles going in the x2 direction and the DNA 18 strand is strongly retarded . CHROMATOGRAPHIC CONDITIONS This behavior can be analytically treated by As mentioned earlier, slalom chromatography introducing the notion of the reorientation time requires a simple chromatographic system needed for macromolecules. The reorientation consisting of a column for gel permeation with time Ί0 is defined as the time needed for a chain an ordinary HPLC system. completely aligned along a direction to completely reorient itself and go in a new Stationary phases direction. Smaller molecules need less time for 69 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 In slalom chromatography, the chromatographic using a Tosoh CCPD dual pump and a Tosoh separation column is packed with “slalom UV-8011 detector coupled to a Shimadzu C-R4A chromatography DNA separation particles”. The integrator. The DNA solution was pre-heated at term “slalom chromatography DNA separation 65°C for 5 min and then cooled on ice until particles” refers to any material which is capable injection to prevent binding via cos sites. This of separating DNA fragments by slalom heat-treated DNA was injected through a chromatography. Slalom chromatography Rheodyne 20-~1 injector. When necessary, separation particles can be inert organic columns were heat-controlled (l0-60°C) by polymers, inert inorganic polymers, silica, or placing them, together with the solvent reservoir, cation exchange resin. The only requirement for in a water bath. DNA retardation under different the slalom chromatography DNA separation conditions were compared in terms of relative particles is that they must have little interaction retention time (RRT). with the DNA fragments23. In earlier experiments, commercially available packed  Result: columns for size-exclusion chromatography 1) Capcell-Pak C1: Large DNA molecules were mainly used: for example, have a high tendency to bind to highly 1) TSKG2000SW- TSK gel columns comprising hydrophobic resins (4.5%)26. In addition, of spherical silica or polymeric resins the Capcell-Pak packings consist of 2) Asahipak GS-32024 silicon-coated fine microbeads 5 µm in 3) Macroporous polystyrene gels diameter that are chemically inert and 4) Porous and non-porous packings originally stable even under alkaline conditions. developed for cation-exchange Resistance to alkali is more favorable chromatography25 for DNA separation, because DNA is in  Porous packings e.g. Silica general acid insoluble, and thus, usually  Non-porous packings e.g. dissolved in weak basic solvents such Ethylenedimethacrylate polymer. as Tris-HCl buffer, pH 7.5-8.0. The Therefore recently a wider range of packing packing, having 5 µm particle diameter, materials was examined. The results obtained allowed separation of the 6.6-kbp on columns developed for reversed-phase fragment from the flow-through fraction chromatography were reported. Two types of at a relatively fast flow-rate (e.g., ~1.0 microbeads (Capcell-Pak and Hypersil-3) were ml/min). The chromatograms obtained chosen for this purpose, with the aim of realizing were very similar to those obtained on the mode of separation, i.e., slalom mode and size-exclusion columns packed with also expanding the separation range to smaller AsahipakGS-310 and GS-510 5 µm DNA molecules. The results improved the particles. Four peaks representing the understanding of the actual separation 4.4, 6.6, 9.4 and 23.1-kbph/Hind111 mechanism in slalom chromatography. fragments were eluted in that order, and retardation of the latter three fragments,  Experimental i.e., 6.6, 9.4 and 23.1 kbp, increased Capcell-Pak columns (250 X 4.6 mm I.D.) of CI when a higher flow-rate was applied. (total carbon content including that used for These observations indicate that the silicon coating, 4.5%), Phe (carbon content, separation achieved on the Capcell-Pak 8.1%) types were used. Cl column is based on the slalom mode, 5 types of Hypersil-3 packings which were and not on the hydrophobic-interaction packed into columns (250 X 6 mm I.D.) were mode. also studied. Phe (phenyl, 5.0%), SAS (trimethylsilyl, 2.6% ), CPS (cyanopropyl,4.0%) 2) Capcell-Pak phe: Another Capcell-Pak MOS (dimethyloctyl, 7.0%) and ODS (octadecyl, packing, Capcell-Pak Phe has the same 10%) were obtained. particle diameter of 5 µm, but a significantly higher carbon content  Chromatographic separation (S.l%,including that used for silicon Various sized DNA fragments (l0-40 kbp) were coating). However,like Capcell-Pak Cl, prepared by digestion with the restriction the Phe column was also found to be endonucleases (ApaI, Xh0I and KpnI). useful for slalom chromatography. The Chromatography was performed essentially by Capcell-Pak Phe column also showed 70 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 size and flow-rate dependency under Capcell-Pak packings or the small 3 µm the normal low-salt conditions, packing size of Hypersil packings 27 suggesting that the separation is based accounted for the different results . on the slalom mode. Mobile phase The mobile phase consists of a sodium phosphate salt (0.01 M), EDTA (0.001 M) 3) Hypersil-3 packings mixture at pH 6.8. Acetonitrile is used as a It is of particular interest to utilize the hydrophobicity modifying agent. smallest possible packing materials for applied slalom chromatography, since Compacting agents (CA) to improve previous experiments demonstrated that oligonucleotide separation: packings having particles of 5, 9, 13 and Sometimes there might be a small difference 19 µm diameter resolved DNA between the retention times of circular and linear fragments larger than 6, 9, 13 and 17 DNA molecules. Hence, in order to get a better kbp respectively. This observation selective separation, Compacting Agents i.e. implies that the use of 3-pm particles spermine, spermidine, hexamine cobalt are might allow resolution of even smaller added. Addition of CA led to a DNA precipitation fragments, for instance, 4 kbp. Five into a pellet form. The addition of these cations Hypersil-3 packings having 3 µm i.e. CA to a DNA solution leads first to the diameter particles (i.e., those derivatized precipitation of the DNA and further addition with trimethylsilyl, cyanopropyl, phenyl, resolubalizes the DNA pellet. The amount of dimethyloctyl or octadecyl groups) were compacting agents to resolubalize various DNA used. fragments corresponds approximately to 50 mM However, none of the λ /Hind111 spermidine, 90 mM spermine, 220 mM fragments was recovered from the five hexamine cobalt28. Hypersil columns under the normal In another experiment, the concentration range conditions. The poor recovery of the of compacting agents varying from 500 mM to 1 fragments is due to excessively strong M was studied. 20 mg per litre of the DNA or effective hydrophobic interaction solutions were injected in triplicate. The mobile between the Hypersil3 packings and phase flow-rate varied from 0.02 to 1.5 ml/min. DNA, although Capcell-Pak columns in this series of experiments, the column gave much more satisfactory results temperature was fixed at 100C. In these under the same conditions. Despite the conditions, DNA fragments didn’t precipitated. significantly different carbon contents of Above 0.1 M, the reptation time of DNA the four Hypersil-3 packings, all of them fragment was weaker to be determined with showed almost the same retardations of accurate precision. λ/HindIII fragments in the presence of At the highest mobile phase velocities, the DNA 10% (v/v) acetonitrile. This fact suggests fragment was close to being fully elongated. The that hydrophobic interaction is no longer increasing extension of large DNA fragment 29 significant when a hydrophobic solvent, increased the separation of oligonucleotide . such as acetonitrile, is included in the Spermine addition in the bulk solvent decreased eluting solvent, and the slalom mode the k value (representing the degree of the becomes predominant. oligonucleotide stretching) for both plasmid and Stronger DNA retardation with Capcell- linear DNA fragment i.e. lead to a compaction of Pak Phe than with C1 seems to be linear and circular DNA fragments. Increasing associated with the stronger spermidine concentration in the mobile phase hydrophobicity of the former packing led to a decrease of stretching of circular DNA than the latter. However, all of the fragment ( k decreased with x for plasmid ) but Hypersil-3 packings, differing in carbon not the one of linear DNA fragment ( k value content (2.6-10%) required the addition constant ). In contrary, hexamine cobalt of more than 5% acetonitrile to the compacted only the linear DNA fragment (k eluting solvent for adequate DNA decreased with x only for the linear DNA recovery. This may suggest that either fragment. This CA difference effect can be special features of the silicon coating of 71 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 useful to separate linear and circular DNA GS-310. Apparently, retardation of each circular 30 fragments and plasmids . DNA became larger when smaller packings were used. The use of larger packings had little FACTORS AFFECTING SLALOM practical merit because most of the circular CHROMATOGRAPHY DNAs were not fully resolved from the fraction. In case of slalom chromatography, the The extent of retardation of a circular form is importance of the presence of narrow open comparable to that of a linear form having half of spaces constructed by packaging microbeads (< the molecular size of the former. The elution 10 µm) developed for high-performance liquid profiles of four circular DNAs with those of linear chromatography (HPLC) and application of a DNA fragments, i.e., 10, 15, 17, 20, and 23 kbp relatively fast flow (0.3 ml/min) were derived from phage DNA. Evidently, the emphasized. Studies showed that none of the (i) obtained curves were very similar for the chemical nature (silica or synthetic polymer) of following pairs: 10 kbp (L) and 20 kbp (C), 17 packings, (ii) pore size of packing material is kbp (L) and 36 kbp (C), 20 kbp (L) and 42 kbp important for separation in slalom (C). This observation strongly suggests that chromatography. So far, some physicochemical DNA separation in slalom chromatography is factors closely related to hydrodynamics have based on extended ‘‘length’’, not on molecular been shown to have a critical effect on the ‘‘mass’’. These observations indicate that super- separation, i.e. coiled circular DNAs take a relatively rigid 1) DNA topology conformation compared with linear forms having 2) Temperature half of the molecular size of the former, probably 3) Flow rate and mobile phase viscosity because the former are stabilised by super-coil 4) Particle size of packing material formation31. Effect of DNA topology Effect of temperature In order to establish the concept that slalom Column temperature is one of the main chromatography is based on a hydrodynamic parameters which could influence DNA principle rather than an equilibrium one, the separation. In order to gain further insight into elution of DNAs having the same molecular the behaviour of DNA in a hydrodynamic flux weights but different topologies (i.e., linear and and enhance the efficiency of the technique, the circular forms) was compared. retention of DNA fragments on a C1 stationary phase was analysed over a wide range of  RESULT column temperature (3-600C). Both linear and circular forms of DNAs having different sizes (approximately 20, 28, 36, and 42  Result kbp) were prepared, and applied to AsahipakS- The retention time values for the 17.05 and29.95 310 columns packed with various sizes of kb fragments (tR) and for the 1.50 kb fragment packing particles (5, 9, 13 and 19 µm in which corresponded to the void volume marker diameter). Extents of their retardations were (tNR) were obtained at various column compared in terms of RRT. Examples of elution temperatures. From the tR and tNR values, the profiles of circular (95%, super-coiled) and linear experimental RRT were calculated for the DNAs are shown in Figure 3. different chromatographic conditions. In this experiment, both circular and linear forms The RRT values increased when T decreased. were co-injected into a column of Asahipak GS- This result confirmed that the temperature acted 310 (particle size, 9 µm) and were eluted at a on the DNA behaviour via two effects: The RRT flow rate of 0.6 ml/min. In each case, circular (C) value varied with the column temperature (T7 forms were eluted much faster than linear (L) function) more strongly that with the linear forms; reasonable to speculate that the extent of velocity. Lower the column temperature, greater retardation i.e., in terms of RRT 0.98 (20 kbp, the separation between non retained and C), 1.25 (20 kbp, L), 1.05 (28 kbp, C), 1.56 (28 retained molecules. Thus, the optimal conditions kbp, L). However, circular forms were also size- for the best separations between the void DNA fractionated, as were linear forms, in a size and fraction and other DNA fragments were flow-rate-dependent manner. The effect of represented by the lowest value of the column particle size of the packings was also examined temperature at a constant flow rate which was by using 5, 9, 13, and 19 particles of Asahipak compatible with a practicable back pressure and 72 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 the prevention of the physical degradation of practicable back pressure and the prevention of 32 33 DNA fragments . the physical degradation of DNA fragments . Effect of flow rate and mobile phase Effect of particle size of packing material viscosity In one of the experiments, DNA fragments Since in slalom chromatography, DNA ranging from 10 to 38 kbp were separated by 2 fragments move further in the direction of columns differing in particle size only. One was laminar flow of the mobile phase, the flow rate v packed with 5 µm particles and the other with 9 and mobile phase viscosity η were some of the µm particles. Both columns separated the main parameters which could influence DNA fragments in the order of smaller to larger. separation. In order to gain further insight into Although the pore sizes are the same, the the fractionation mechanism and enhance the column packed with 5 µm particles was superior efficiency of the technique, retention of DNA for the separation of smaller fragments(less than fragments on a stationary phase C1was 20 kbp), while larger fragments (greater than 20 analyzed over a wide range of glycerol kbp), were better separated by the column concentrations (used as a viscosity modifier at packed with 9 µm particles. This result indicated concentrations of 0 to 1 M) and at various linear that the fractionation range depends on the velocities v (i.e. flow rates values varying from particle size of the column packing but not on 0.05 to 1.2ml/min). the pore size. In another experiment using 4 columns (average  Results particle diameters 5, 9, 13.1, 19.1 µm) the RRT Recoveries of the DNA fragments were was plotted against DNA length i.e. no. of calculated from the chromatographic areas. The basepairs. These 4 columns showed different DNA recovery at constant flow-rate was globally ranges of resolution: e.g.at a flow rate of 0.6 identical (difference <10%) whatever the value ml/min, the 5, 9, 13.1, 19.1 µm columns could of glycerol concentration in the eluent. Thus, it separate DNA fragments greater than 7, 9, 13, can be concluded that the glycerol effect on 17 kbp, respectively. Smaller packings showed relative retention time (RRT) values of the DNA better resolution for smaller DNA fragments, fragments was the result of the change in the whereas larger ones were better for larger mobile-phase viscosity. Also, this showed that fragments8,10. the destabilizing effect of polyol on DNA related to its capacity to interact with the polynucleotide ADVANTAGES OF SLALOM solvation sites did not significantly affect the CHROMATOGRAPHY fragment integrity over the glycerol At present, it is the only chromatographic concentration range studied. method applicable to the size-dependent The parameters η and v acted in the same separation of large DNA molecules. It is unique manner on DNA retardation (increase in RRT because it is based on the hydrodynamic value). The main difference between these two principle that the mobility of fibrous molecules in factors was that the viscosity increase was a column is determined by their length and truly associated with a concomitant enhancement of size-dependent separation occurs. the analysis time, while the flow-rate increase Its advantages are summarized below: was associated with the decrease in the analysis 1) Both preparative and analytical uses are time. Higher the liquid velocity, greater is the possible. As an analytical procedure, it separation between non-retained and retained provides information on the length of molecules and shorter the analysis time. This polynucleotides. fact shows the advantage of the slalom 2) The experiment procedure is very chromatography principle on the equilibrium simple and rapid. Ordinary HPLC principle of the classical chromatographic apparatus and a gel permeation column modes. are sufficient as equipment. Thus, the optimal conditions for the best 3) Results can be easily predicted. separations between the void DNA fraction and 4) Only isocratic elution programme is other DNA fragments were represented by the necessary and there is no need for highest values of the linear velocity at a constant column washing or reequilibration. viscosity which was compatible with a 5) The separation and recovery of DNA fragments are very rapid in comparison 73 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 with gel electrophoresis. Recovered applied to biomolecules which are based on DNA fragments are free of undesirable equilibrium phenomenon, this technique is contamination originating from the based on a completely different principle of agarose gel. hydrodynamic phenomenon. The column 6) DNA can be detected without the use of packing serves only for the construction of a harmful reagent, such as ethidium spaces through which DNA passes. Hence this bromide. technique is important from the viewpoint of 7) This procedure provides a new effective study of physiochemistry of macromolecules. tool for physicochemical and Theoretical background of slalom hydrodynamic studies of DNA. chromatography is still giving its first steps therefore further investigations in this field are required to expand the horizons of slalom chromatography. LIMITATIONS OF SLALOM CHROMATOGRAPHY Table 1: Comparison of chromatographic 1) At present, the range of separable sizes for modes available for size fractionation of DNA molecules is not wide; fragments of 5- nucleic acids 50 kbp are separable using commercially Mode Size range for Speed Resolution available packing particles. However further D.S. DNA (kbp) Ion-exchange < 25 High High extensive studies will improve the situation. Gel-permeation <6 Low Low 2) The resolution efficiency is still inferior to Gel electrophoresis - Low High that of gel electrophoresis. Slalom 5-50 High Medium 3) The flow-rate must be relatively high. This may cause physical degradation of extremely large DNA fragments. However DNA fragments less than 50 kbp proved to be generally very stable under the conditions of most experiments (e.g. flow- rates less than 1.2 ml/min). APPLICATIONS This principle will provide with a valuable tool for nucleic acid research. Although only application to DNA have been reported so far, it should be also useful for RNA research. Some possible applications are listed below: 1) Size-dependent separation of DNA 2) Estimation of size of DNA 3) Monitoring and analysis of size change of DNA 4) Separation of DNA and RNA based on conformation or topology 5) Analysis of interaction of DNA with other molecules, such as DNA binding proteins 6) Distinction of types of circular DNA, e.g. Fig. 1: Illustration of DNA separation in super-coil, relaxed and single strand slalom chromatography 7) Studies of the physicochemical properties of nucleic acids, e.g. rigidity, elasticity, bendability etc. 8) Hydrodynamic studies of nucleic acids6. CONCLUSION Slalom chromatography provides us with a valuable tool for nucleic acid research. Unlike other chromatographic separation modes 74 IJRPC 2012, 2(1) Aditi Chitnis et al ISSN: 22312781 in solution studied by fluorescence microscopy. Cold Spring Harb Symp Quant Biol. 1983;47 Pt 1:177-187. 3. Ausserer WA and Biros ML. High- resolution analysis and purification of synthetic oligonucleotides with strong anion-exchange HPLC. Biotechniques. 1995;19:136-139. 4. Gilar M, Fountain KJ, Budman Y, Neue UD, Yardley KR, Rainville PD et al. Ion- pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: Retention prediction. J Chromatogr A. 2002 Jun 7;958(1- 2):167-182. 5. Crowther JB, Jones R and Hartwick RA. High performance liquid Fig. 2: Representation of the progression of chromatography of the oligonucleotides. the DNA chains (arrows) through the closed J Chromatogr. 1981;217:479-490. column packing particles 6. Kasai K. Size-dependent chromatographic separation of nucleic acids. J Chromatogr. 1993 Aug 25;618(1-2):203-221. 7. Boyes BE, Walker DG and McGeer PL. Separation of large DNA restriction fragments on a size-exclusion column by a nonideal mechanism. Anal Biochem. 1988;170:127-134. 8. Hiarabayashi J and Kasai K. 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