Fig 11.Measuremet for detemnng e w12 Volume The height equivalent to a theoretical plate.H.is given by H=UN where L is the column length and N is the number of theoretical plates. Any parameter change that increases N will also increase Rs.The relationship eee 100 to625 will improve the esotioan resolution by a factor of 2.5.rather than efficiency (N)is particle size and the colu edure ted tha acking proc colu ss than 10m are commercially available only in pre-packed formats. nn. 16
16 The number of theoretical plates is sometimes reported as plates per metre of column length (N/L). The height equivalent to a theoretical plate, H, is given by H = L/N where L is the column length and N is the number of theoretical plates. Any parameter change that increases N will also increase Rs. The relationship between the two is defined by the square root of N. For example, an increase in N from 100 to 625 will improve the resolution by a factor of 2.5, rather than 6.25. The main contribution to column efficiency (N) is particle size and the efficacy of the column packing procedure. It should be noted that the smaller the particle size, the more difficult it is to pack an efficient column. This is the reason why reversed phase media with particle sizes less than 10 µm are commercially available only in pre-packed formats. v1 w1/2 Peak height Volume Abs Fig. 11. Measurements for determining column efficiency. V1 is the retention volume of the peak and W1/2 is the peak width (volume) at half peak height
Selectivity n of the solute peaks and.unlike ency.depends strongly on the chemical properties of the chromatography medium. The selectivity,o.for two peaks is given by a=k,'k'=V2VV-V。=VV, where V,and V,are the retention volumes,and k,'/k,'are the capacity factors for peaks 1 and 2 respectively,and V is the void volume of the column. Selectivity is affected by the surface chemistry of the reversed phase medium,the nature and composition of the mobile phase and the gradient shape Fig 12.Selectivit rison betwe .Upp a)and e)Sephasi Protein C 5 jm 4. d)and h 5%aceton SATI(ORVYIIHPLFH) Protein C4 s Peptide C8 asl Peptide C18 RPC C2/C18 b 50005200 0050005020250m 50t5●0035m 17
17 Selectivity Selectivity (α) is equivalent to the relative retention of the solute peaks and, unlike efficiency, depends strongly on the chemical properties of the chromatography medium. The selectivity, α, for two peaks is given by α = k2 ´ /k1 ´ = V2 - V0 /V1 – V0 = V2 /V1 where V1 and V2 are the retention volumes, and k2 ´ /k1 ´ are the capacity factors, for peaks 1 and 2 respectively, and V0 is the void volume of the column. Selectivity is affected by the surface chemistry of the reversed phase medium, the nature and composition of the mobile phase, and the gradient shape. Fig. 12. Selectivity comparison between different silica based media at pH 2.0 and pH 6.5. A mixture of closely related angiotensin peptides was used as sample. (Work by Amersham Pharmacia Biotech AB, Uppsala, Sweden.) pH 2 a) b) c) d) e) f) g) h) pH 2 pH 2 pH 2 pH 6.5 pH 6.5 pH 6.5 pH 6.5 1 2+3 4 5+6 7+8 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 1 2 34 5+6 7+8 1 2 3 4 5+6 7+8 1 2 4 3 6 7+8 5 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 1 2 3+4 6 5 8 7 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 0.0 5.0 10.0 15.0 20.0 25.0 min 1 2 34 6 5 8 7 1 2 6 4 3 5 8 7 1 2 3 4 5+6 7+8 Sephasil Protein C4 Sephasil Peptide C8 Sephasil Peptide C18 µRPC C2/C18 1. Val4-lle7-AT III (RVYVHPI) 2. Ile7-AT III (RVYIHPI) 3. Val4-AT III (RVYVHPF) 4. Sar1-Leuß-AT II (Sar-RVYIHPL) (Sar=sarcosine, N-methylglycine) 5. AT III (RVYIHPF) 6. AT II (DRVYIHPF) 7. des-Asp1-AT I (RVYIHPLFHL) 8. AT I (DRVYIHPFHL) Columns: a) and e) Sephasil Protein C4 5 µm 4.6/100 b) and f) Sephasil Peptide C8 5 µm 4.6/100 c) and g) Sephasil Peptide C18 5 µm 4.6/100 d) and h) µRPC C2/C18 ST 4.6/100 Eluent A (pH 2): 0.065% TFA in distilled water Eluent B (pH 2): 0.05% TFA, 75% acetonitrile Eluent A (pH 6.5): 10 mM phosphate Eluent B (pH 6.5): 10 mM phosphate, 75% acetonitrile Flow: 1 ml/min System: ÄKTApurifier Gradient: 5–95% B in 20 column volumes
Good selectivity Poor selectivity 2aenoy cency Fig.13.The effect of selectivity and efficiency on resolution. Both high column efficiency and good selectivity are important to overall resolution.However.changing the selectivity in a chromatographic experiment is easier than changing the efficiency.Selectivity can be changed by changing easily modified conditions like mobile phase composition or gradient shape Binding capacity The available binding capacity of a reversed phase medium is a quantitative measure of its ability to adsorb solute molecules under static conditions.The dynamic binding capacity is a measure of the available binding capacity at a specific flow rate.Both values are extremely important for preparative work n of solte hich ind toepen oype oportional to the depend on the the pr chemical perimental conditionsed and physical properti of the versed phase medium (porosity.etc.)and The porosity of the bead is an important factor which influences binding capacity.The entire hydrophobic surface of macroporous media is available for binding solute.Large solute molecules(i.e.high molecular weight)may be excluded from media of smaller pore size and only a small fraction of the whole hydrophobic surface will be used.When maximum binding capacity is required,a medium with pores large enough to allow all the molecules of interest to enter freely must be used. 18
18 Fig. 13. The effect of selectivity and efficiency on resolution. Both high column efficiency and good selectivity are important to overall resolution. However, changing the selectivity in a chromatographic experiment is easier than changing the efficiency. Selectivity can be changed by changing easily modified conditions like mobile phase composition or gradient shape. Binding capacity The available binding capacity of a reversed phase medium is a quantitative measure of its ability to adsorb solute molecules under static conditions. The dynamic binding capacity is a measure of the available binding capacity at a specific flow rate. Both values are extremely important for preparative work. The amount of solute which will bind to a medium is proportional to the concentration of immobilised ligand on the medium and also depends on the type of solute molecule being adsorbed to the medium. The available and dynamic binding capacities depend on the specific chemical and physical properties of the solute molecule, the properties of the reversed phase medium (porosity, etc.) and the experimental conditions during binding. The porosity of the bead is an important factor which influences binding capacity. The entire hydrophobic surface of macroporous media is available for binding solute. Large solute molecules (i.e. high molecular weight) may be excluded from media of smaller pore size and only a small fraction of the whole hydrophobic surface will be used. When maximum binding capacity is required, a medium with pores large enough to allow all the molecules of interest to enter freely must be used. High efficiency Low efficiency High efficiency Low efficiency Good selectivity Poor selectivity
Critical parameters in reversed phase chromatography Column length olution of high molecular weight biomolecules in reversedp separations is gth than is t of small organi oteins.large peptides an he ds nucleic ac may be purified effectively on short columns and Increasing co umn length d not Improve resolution significantly.The resolution of small peptides(including some peptide digests)may sometimes be improved by increasing column length.For example the number of peaks detected when a tryptic digest of carboxamidomethylated transferrin was fractionated by RPC increased from 87 on a 5 cm long column to 115 on a 15 cm long column and 121 on a 25 cm long column (6). The partition coefficients of high molecular weight solutes are very sensitive to small chang s in mobile phase composition and hence lar rge molecules desorb in a rganic ntration Th retention behaviour of d to b cha s may ed by n in partiti ent to c ol ength.When Smal change er concentration result i I changes in the partition coefficient,longer column lengths increase resolution The use of gradient elution further reduces the significance of column length for the resolution of large biomolecules by reversed phase chromatography.Gradients are required since most biological samples are complex mixtures of molecules that vary greatly in their adsorption to the reversed phase medium.Due to this variety of adsorption affinities,the mobile phase must have a broad range of eluting power to ensure elution of all the bound solute molecules.Under these conditions,especially with moderate to steep gradient slopes,column length is not a critical factor with regard to resolution Flow rate Flow rate is expected to be an important factor for resolution of small molecules including small peptides and protein digests,in reversed phase separations. However,reversed phase chromatography of larger biomolecules,such as proteins and recombinantly produced peptides.ai pears to be insensitive to flow rate.In fact.low flow rate sed with lc ns,may actually decrease resolution du e to in ased lon sion of the solute molecules as they traverse the length of the e columr The flow rate used during the loading of the nple solution is especially signific large scale preparative reversed phas chromatography,a though not critical du ng analytical experiments.Dynamic binding capacity will vary depending on the flow rate used during sample loading.When scaling up a purification,the dynamic binding capacity should be determined in order to 19
19 Critical parameters in reversed phase chromatography Column length The resolution of high molecular weight biomolecules in reversed phase separations is less sensitive to column length than is the resolution of small organic molecules. Proteins, large peptides and nucleic acids may be purified effectively on short columns and increasing column length does not improve resolution significantly. The resolution of small peptides (including some peptide digests) may sometimes be improved by increasing column length. For example, the number of peaks detected when a tryptic digest of carboxamidomethylated transferrin was fractionated by RPC increased from 87 on a 5 cm long column to 115 on a 15 cm long column and 121 on a 25 cm long column (6). The partition coefficients of high molecular weight solutes are very sensitive to small changes in mobile phase composition and hence large molecules desorb in a very narrow range of organic modifier concentration. The retention behaviour of large molecules may be considered to be governed by an on/off mechanism (i.e. a large change in partition coefficient) which is insensitive to column length. When small changes in organic modifier concentration result in small changes in the partition coefficient, longer column lengths increase resolution. The use of gradient elution further reduces the significance of column length for the resolution of large biomolecules by reversed phase chromatography. Gradients are required since most biological samples are complex mixtures of molecules that vary greatly in their adsorption to the reversed phase medium. Due to this variety of adsorption affinities, the mobile phase must have a broad range of eluting power to ensure elution of all the bound solute molecules. Under these conditions, especially with moderate to steep gradient slopes, column length is not a critical factor with regard to resolution. Flow rate Flow rate is expected to be an important factor for resolution of small molecules, including small peptides and protein digests, in reversed phase separations. However, reversed phase chromatography of larger biomolecules, such as proteins and recombinantly produced peptides, appears to be insensitive to flow rate. In fact, low flow rates, typically used with long columns, may actually decrease resolution due to increased longitudinal diffusion of the solute molecules as they traverse the length of the column. The flow rate used during the loading of the sample solution is especially significant in large scale preparative reversed phase chromatography, although not critical during analytical experiments. Dynamic binding capacity will vary depending on the flow rate used during sample loading. When scaling up a purification, the dynamic binding capacity should be determined in order to
timum flov ng the reflects the kinetics of the so ding proce efficiency of this step can have enormous consequences for the results of a large scale preparative purification. Temperature Temperature can have a profound effect on reversed phase chromatography especially for weight solute uch as short he viscosi f the e mobil peptide used in reversed phase chromatography de reases rature.S nce mass transport of sol te between the mobile phase and the stationary phase is a diffusion-controlled process. decreasing solvent viscosity generally leads to more efficient mass transfer and,therefore,higher resolution.Increasing the temperature of a reversed phase column is particularly effective for low molecular weight solutes since they are suitably stable at the elevated temperatures. Mobile phase In many cases.the colloquial term used for the mobile phases in reversed phase chromatography is"buffer".However,there is little buffering capacity in the mobile phase solutions since they usually contain strong acids at low pH with large concentrations of organic solvents.Adequate buffering capacity should be maintained when working closer to physiological conditions. Organic solvent The organic solvent(modifier)is added to lower the polarity of the aqueous mobile phase.The low er the polarity of the mobile phase the eater its eluting strength in rever ed phase chr 8 gh a large of organic can be reversed ph ide romatography. tice ed.TI e two n are aceto althoug e more popu lar choice. oprop propanol can be employed be e of its strong eluti g propertie by its high viscosity which results in lower column efficiencies and higher back- pressures.Both acetonitrile and methanol are less viscous than isopropanol. All three solvents are essentially UV transparent.This is a crucial property for reversed phase chromatography since column elution is typically monitored using UV detectors.Acetonitrile is used almost exclusively when separating peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum (typically less than 225 nm)and acetonitrilep provides much lower background orbance than other co mon solvents at lo wavelengths. 20
20 assess the optimum flow rate for loading the sample. Dynamic binding capacity is a property of the gel that reflects the kinetics of the solute binding process. The efficiency of this step can have enormous consequences for the results of a large scale preparative purification. Temperature Temperature can have a profound effect on reversed phase chromatography, especially for low molecular weight solutes such as short peptides and oligonucleotides. The viscosity of the mobile phase used in reversed phase chromatography decreases with increasing column temperature. Since mass transport of solute between the mobile phase and the stationary phase is a diffusion-controlled process, decreasing solvent viscosity generally leads to more efficient mass transfer and, therefore, higher resolution. Increasing the temperature of a reversed phase column is particularly effective for low molecular weight solutes since they are suitably stable at the elevated temperatures. Mobile phase In many cases, the colloquial term used for the mobile phases in reversed phase chromatography is “buffer”. However, there is little buffering capacity in the mobile phase solutions since they usually contain strong acids at low pH with large concentrations of organic solvents. Adequate buffering capacity should be maintained when working closer to physiological conditions. Organic solvent The organic solvent (modifier) is added to lower the polarity of the aqueous mobile phase. The lower the polarity of the mobile phase, the greater its eluting strength in reversed phase chromatography. Although a large variety of organic solvents can be used in reversed phase chromatography, in practice only a few are routinely employed. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice. Isopropanol (2- propanol) can be employed because of its strong eluting properties, but is limited by its high viscosity which results in lower column efficiencies and higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol. All three solvents are essentially UV transparent. This is a crucial property for reversed phase chromatography since column elution is typically monitored using UV detectors. Acetonitrile is used almost exclusively when separating peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum (typically less than 225 nm) and acetonitrile provides much lower background absorbance than other common solvents at low wavelengths