N5to0eoa 8e Restricted mass transfer More efficient mass transfer The surface of the polystyrene bead is itself strongly hydrophobic and.therefore. when left underivatised unlike silica gels that have hydrophobic ligands grafted to a hydrophilic surface. The porosity of the reversed phase beads is a crucial factor in determining the available capacity for solute binding by the medium.Note that this is not the capacity factor (k)but the actual binding capacity of the medium itself.Media with p sizes of ar oloules and prpudes Medla bdin of recombinant p eptidesand roteins that can withstand the stringent conditions of reversed phase chromatography. The ligands The selectivity of the reversed phase medium is predominantly a function of the speaking,linear ular liga nds used in phase applications. e typical hydrocar ds are e shown in 8glatiocatoagantWTmocatbaaeapngoap.oas1gead CHg-CHa -0 C-C-C-CC 11
11 Fig. 5. Reverse phase media with wide pores allow the most efficient transfer of large solute molecules between the mobile and the stationary phases. The surface of the polystyrene bead is itself strongly hydrophobic and, therefore, when left underivatised unlike silica gels that have hydrophobic ligands grafted to a hydrophilic surface. The porosity of the reversed phase beads is a crucial factor in determining the available capacity for solute binding by the medium. Note that this is not the capacity factor (k´) but the actual binding capacity of the medium itself. Media with pore sizes of approximately 100 Å are used predominately for small organic molecules and peptides. Media with pore sizes of 300 Å or greater can be used in the purification of recombinant peptides and proteins that can withstand the stringent conditions of reversed phase chromatography. The ligands The selectivity of the reversed phase medium is predominantly a function of the type of ligand grafted to the surface of the medium. Generally speaking, linear hydrocarbon chains (n-alkyl groups) are the most popular ligands used in reversed phase applications. Some typical hydrocarbon ligands are shown in Figure 6. Fig. 6. Typical n-alkyl hydrocarbon ligands. (A) Two-carbon capping group, (B) Octyl ligand, (C) Octadecyl ligand. Restricted mass transfer More efficient mass transfer 50–100 Å Narrow pore 300 Å Wide pore —O—Si—CH2—CH3 CH3 CH3 —O—Si—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH3 CH3 CH3 —O—Si—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH2—CH CH3 CH3 (A) (B) (C)
Although it is pot posible to predict theorecal which ligand will be best for a particu h is:the m hydroph molecul eds to be The more hydrophilic molecules tend to require strongly hydrophobic immobi Iligands in order to achieve sufficient binding for separation.Typically,chemically synthesised peptides and oligonucleotides are efficiently purified on the more hydrophobic C18 ligands.Proteins and recombinant peptides.because of their size.behave as hydrophobic molecules and most often bind very strongly to C18 ligands.They are usually better separated on C8 ligands.The less hydrophobic eight carbon alkyl chain is less disruptive to the protein and peptide structures since lower concentrations of orga ic solvent are required for elution.In addition to ligand structure,the density of the immobilised hydrocarbon ligands on the rfac also influence e selectivity shown by sed phas media repr mical de of th ritical for reversed phase ch atography with con istent batch-to-batch selectivit The hydrocarbon ligands are generally silica gel via silanol groups on the silica surface using chlorotrialkylsilane reagents.Not all of the silanol groups will be substituted in this coupling reaction.The C18 and C8 reagents are large and bulky so that steric hindrance often prevents complete derivatisation of all the available silanol groups.The residual silanol groups are believed to be responsible for the deleterious mixed mode ion exchange effects often present during reversed phase separation of biomolecules.In order to reduce these damaging side effects.the residual silanol groups are reacted with smaller e reagents(chlorotrimethyl-and chlo otriethylsilanes)where steric effects do not interfere with complete coverage of the silanol groups remaining on rfac ref erred to as“eng ing".The so repro step is well be ved reversed phase medium. reaction is shown in Figure7 The de y ctadecyl chains by reaction with CHa CHa -Si-OH Cl-S(CH2)17-CH3-Si-O-Si-(CH2)17-CH3 HC CHa The particle size of the bead,as measured by its diameter, has important packand fo he y of the The ane pucee bed which can be usefully generally used for large scale preparative and process applications due to their increased capacity and lower pressure requirements at high flow rates.Small scale preparative and analytical separations use smaller particles since separation efficiency,i.e.peak width,is directly related to particle size(see section on 12
12 Although it is not possible to predict theoretically which ligand will be best for a particular application, a good rule of thumb is: the more hydrophobic the molecule to be purified, the less hydrophobic the ligand needs to be. The more hydrophilic molecules tend to require strongly hydrophobic immobilised ligands in order to achieve sufficient binding for separation. Typically, chemically synthesised peptides and oligonucleotides are efficiently purified on the more hydrophobic C18 ligands. Proteins and recombinant peptides, because of their size, behave as hydrophobic molecules and most often bind very strongly to C18 ligands. They are usually better separated on C8 ligands. The less hydrophobic eight carbon alkyl chain is less disruptive to the protein and peptide structures since lower concentrations of organic solvent are required for elution. In addition to ligand structure, the density of the immobilised hydrocarbon ligands on the silica surface also influences the selectivity shown by reversed phase media. Therefore, reproducible chemical derivatisation of the silica surface is critical for efficient reversed phase chromatography with consistent batch-to-batch selectivity. The hydrocarbon ligands are generally coupled to the silica gel via silanol groups on the silica surface using chlorotrialkylsilane reagents. Not all of the silanol groups will be substituted in this coupling reaction. The C18 and C8 reagents are large and bulky so that steric hindrance often prevents complete derivatisation of all the available silanol groups. The residual silanol groups are believed to be responsible for the deleterious mixed mode ion exchange effects often present during reversed phase separation of biomolecules. In order to reduce these damaging side effects, the residual silanol groups are reacted with smaller alkylsilane reagents (chlorotrimethyl- and chlorotriethylsilanes) where steric effects do not interfere with complete coverage of the silanol groups remaining on the surface of the silica gel. This process is referred to as “end-capping”. The extent of end-capping also affects selectivity, so reproducibility in the capping step is critical for a well behaved reversed phase medium. The derivatisation reaction is shown in Figure 7. Fig. 7. Substitution of silica with octadecyl chains by reaction with monochlorodimethyloctadecylsilane. The particle size of the bead, as measured by its diameter, has important consequences for the size of the chromatographic bed which can be usefully packed, and for the efficiency of the separation. The larger particle size media are generally used for large scale preparative and process applications due to their increased capacity and lower pressure requirements at high flow rates. Small scale preparative and analytical separations use smaller particles since separation efficiency, i.e. peak width, is directly related to particle size (see section on —Si—OH + Cl—Si—(CH2)17—CH3 —Si—O—Si—(CH2)17—CH3 + HCl CH3 CH3 CH3 CH3
eemintio of the eo veen two peaks 2 R) efficiency and selectivity).Analytical and small scale preparative usually performed with 3 and 5 um beads while larger scale preparative applications(pilot and process scale)are usually performed with particle sizes of 15 Hm and greater.Micropreparative and small scale preparative work can be accomplished using particle sizes of 3 um since the limited capacity of the small columns packed with these media is not a severe problem when only small quantities of material are purified. Resolution in reversed phase chromatography Resolution Adequate resolution and recovery of purified biological material is the ultimate defined as the dist oen the con e ntion time or volum e divided by the a in peaks as mea red by g vidth the xamp an Rs value of 1.0 in 6 purity ha ectve pe. ved 100%puriy and ruires an Bs alue reater than 15 FIe.9 a recovery). aseline rese on Calculating Rs is the simplest method for quantitating the actual separation achieved between two solute molecules.This simple relationship can be expanded to demonstrate the connection between resolution and three fundamental parameters of a chromatographic separation.The parameters have been derived from chromatographic models based on isocratic elution but are still appropriate when used to describe their effects on resolution when discussing gradient elution (consider a continuous linear gradient elution to be a series of small isocratic elution steps).The nar ameters that contribute to peak resolution are column selectivity.column lency and the column ret ention factor 0
13 efficiency and selectivity). Analytical and small scale preparative applications are usually performed with 3 and 5 µm beads while larger scale preparative applications (pilot and process scale) are usually performed with particle sizes of 15 µm and greater. Micropreparative and small scale preparative work can be accomplished using particle sizes of 3 µm since the limited capacity of the small columns packed with these media is not a severe problem when only small quantities of material are purified. Resolution in reversed phase chromatography Resolution Adequate resolution and recovery of purified biological material is the ultimate goal for reversed phase preparative chromatography. Resolution, Rs, is generally defined as the distance between the centres of two eluting peaks as measured by retention time or volume divided by the average width of the respective peaks (Fig. 8). For example, an Rs value of 1.0 indicates 98% purity has been achieved (assuming 98% peak recovery). Baseline resolution between two well formed peaks indicates 100% purity and requires an Rs value greater than 1.5 (Fig. 9). Calculating Rs is the simplest method for quantitating the actual separation achieved between two solute molecules. This simple relationship can be expanded to demonstrate the connection between resolution and three fundamental parameters of a chromatographic separation. The parameters have been derived from chromatographic models based on isocratic elution but are still appropriate when used to describe their effects on resolution when discussing gradient elution (consider a continuous linear gradient elution to be a series of small isocratic elution steps). The parameters that contribute to peak resolution are column selectivity, column efficiency and the column retention factor. Fig. 8. Determination of the resolution between two peaks. v2 v1 w1 w2 v2–v1 (w2 + w1)/2 Rs=
Fig.9.Separation results with different resoluti ) 4 Resolution Rs is a function of selectivity o.efficiency (number of theoretical plates N)and the average retention factor,k',for peaks 1 and 2. Capacity factor The capacity factor,k.is related to the retention time and is a reflection of the proportio me a particular so uteresides in n the stationary pha to the mobile phase.Long retention times result in large values of k.The capacity factor is not the same as the available binding capacity which refers to the mass of the solute that a specified amount of medium is capable of binding under defined conditions.The capacity factor,k,can be calculated for every peak defined in a chromatogram.using the following equations. Capacity factor-kmole in stationary phase moles of solute in mobile phase k'=TR-To VR-Vo T。 V。 where Tgand Ve are the retention time and retention volume.respectively,of the solute,and T and V the retention time and retention volume,respectively,of an unretarded solute. 14
14 Fig. 9. Separation results with different resolution. 98% B A B 98% A 98% B Rs=1 A B 100% A 100% B Rs=1.5 Resolution Rs is a function of selectivity α, efficiency (number of theoretical plates N) and the average retention factor, k´, for peaks 1 and 2. Capacity factor The capacity factor, k´, is related to the retention time and is a reflection of the proportion of time a particular solute resides in the stationary phase as opposed to the mobile phase. Long retention times result in large values of k´. The capacity factor is not the same as the available binding capacity which refers to the mass of the solute that a specified amount of medium is capable of binding under defined conditions. The capacity factor, k´, can be calculated for every peak defined in a chromatogram, using the following equations. Capacity factor=k´= TR – TO VR – VO TO VO where TR and VR are the retention time and retention volume, respectively, of the solute, and To and Vo the retention time and retention volume, respectively, of an unretarded solute. moles of solute in stationary phase moles of solute in mobile phase k´= Rs= 1 (α-1) 4 α ( N ) k´ 1 + k
respectively. V1- V2 3 In the resolution equation previously described,the k'value is the average of the capacity factors of the two peaks being resolved.Unlike non-adsorptive chromatographic methods (e.g.gel filtration).reversed phase chromatography can have very high capacity factors.This is because the experimental conditions can be chosen to result in peak retention times greatly in excess of the total column volume. Efficiency (N) The efficiency of a packed column is expressed by the number of theoretical plates.N.N is a dimensionless number and reflects the kinetics of the chromatographic retention mechanism.Efficiency depends primarily on the physical properties of the chromatographic medium together with the chromatography column and system dimensions.The efficiency can be altered by changing the cle size.the colimn length,or the flow rate.The expression of theoretical plates"is an archaic term carried over fro oretical mparis n of a ch apparatus c greater of th ography colu mn ucy and dingly.the h e grea The coum fficiency (N)can be determined empirically using the equation pelow based upon the zone broadening that occurs when a solute molecule is eluted from the column (Fig.11). The number of theoretical plates.N.is given by N=5.54VW)2 where V,is the retention volume of the peak and W is the peak width(volume) at half peak height. 6
15 In the resolution equation previously described, the k´ value is the average of the capacity factors of the two peaks being resolved. Unlike non-adsorptive chromatographic methods (e.g. gel filtration), reversed phase chromatography can have very high capacity factors. This is because the experimental conditions can be chosen to result in peak retention times greatly in excess of the total column volume. Efficiency (N) The efficiency of a packed column is expressed by the number of theoretical plates, N. N is a dimensionless number and reflects the kinetics of the chromatographic retention mechanism. Efficiency depends primarily on the physical properties of the chromatographic medium together with the chromatography column and system dimensions. The efficiency can be altered by changing the particle size, the column length, or the flow rate. The expression “number of theoretical plates” is an archaic term carried over from the theoretical comparison of a chromatography column to a distillation apparatus. The greater the number of theoretical plates a column has, the greater its efficiency and correspondingly, the higher the resolution which can be achieved. The column efficiency (N) can be determined empirically using the equation below based upon the zone broadening that occurs when a solute molecule is eluted from the column (Fig. 11). The number of theoretical plates, N, is given by N = 5.54 (V1 /W1/2) 2 where V1 is the retention volume of the peak and W1/2 is the peak width (volume) at half peak height. Fig. 10. Hypothetical chromatogram. VO=void volume, VC=total volume, V1 , V2, and V3 are the elution (retention) volumes of peaks 1, 2 and 3, respectively. 1 2 3 vc v0 v1 v2 v3