Fig.8.An ele on micrograph of MonoBeads showing their distinet monodispersity. The resolution which can be achieved o any chrom ographic rix is a result oh the ency all tion separations. Scale-up to SOURCE Q and S(see Chapter 5),Q and SP Sepharose High Performance and Qand SP Sepharose Fast Flow (see Chapter 6)is simple,since these gels have similar selectivities to MonoBeads based media. Properties Chemical stability be used with solutions of most buffersused in biochemical of bio molecules and n warer-alcohol (C1C4)and solutions ning and the use of conditions nec samples.An noBeads with organic sol ents is given in 24
The name MonoBeads is derived from the unique monodisperse nature of the matrix. This monodispersity (Fig. 8) was accomplished through a process developed by Professor John Ugelstad of SINTEF, Trondheim, Norway. Fig. 8. An electron micrograph of MonoBeads showing their distinct monodispersity. The resolution which can be achieved on any chromatographic matrix is a result of a combination of the efficiency and selectivity of the system. Maximum efficiency is obtained through the use of small, perfectly spherical, monodisperse particles, optimally packed in a well designed column. All pre-packed MonoBeads columns have efficiencies at about 25 000 plates per metre. High efficiency, coupled with the excellent selectivity of the Q and S substituents, results in high resolution separations. Scale-up to SOURCE Q and S (see Chapter 5), Q and SP Sepharose High Performance and Q and SP Sepharose Fast Flow (see Chapter 6) is simple, since these gels have similar selectivities to MonoBeads based media. Properties Chemical stability The gels are stable for continuous use in the pH range 2-12, although pH values as high as 14 can be used during cleaning and sanitizing procedures. MonoBeads can be used with solutions of most buffers used in biochemical separations of biomolecules and in water-alcohol (C1 - C4) and acetonitrile-water solutions. The resistance of the MonoBeads matrix to organic solvents allows complete cleaning and the use of conditions necessary for the solubilization of very hydrophobic samples. An example of the use of MonoBeads with organic solvents is given in Figure 9, which shows the analysis of the peptide bacitracin on Mono S using lithium chlorate as the eluting salt and 90% methanol as the liquid phase (7). 24
nme ecthde (DMSOs solutions of urea.ethvle separation properties he g the ion hange be Physical stability o流am low rates.As a conse HR these c pumps
Fig. 9. Separation of the peptide bacitracin on Mono S. (Work by Pharmacia Biotech, Uppsala, Sweden.) Dimethylsulphoxide (DMSO) and similar solvents can be used, but will change the separation properties of the gels. Aqueous solutions of urea, ethylene glycol and similar compounds can be used but will increase the back-pressures due to their higher viscosities. Non-ionic detergents, zwitterionic detergents or detergents with the same charge as the ion exchange groups may be used. Oxidizing agents should be avoided. Physical stability MonoBeads are based on highly rigid beads which means that they can be used at high flow rates. As a consequence of the monodisperse nature of the matrix these high flow rates do not result in high back-pressures. For example, an HR 5/5 column (5 mm inner diameter and 50 mm bed height) packed with a MonoBeads matrix normally generates a back-pressure of 1.0-1.5 MPa (10-15 bar) when operated at a flow rate of 1 ml/min (300 cm/h). Note: These back-pressures are beyond the operating limits of standard laboratory peristaltic pumps. W-X? 7R1? ?J@?@L ?7@@@)X? ?@e?@)KO-X?W-K? ?@e?(R4@?,?7R@@@@@@@@@? S@U?@?@@?@@?@?@? ?@KO&?,?3T@@?@@?@?@? ?@@0R+Y?V+R'?@@?@?@? ?@?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@ ?@?@?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?he?@ ?@ @?heJ@ ?@ @?he7@ ?@ @?he@@ ?@ @?he@@ ?@ @?he@@ ?@ @?he@@ ?@ ?J@?he@@ O2@@@@@@@@@@@@@@e ?@ ?7@?he@@ O2@@@@0M ?@ ?@@?he@@ O20M ?@ ?@@?he@@ W20M ?@ ?@@?he@@ ?O.M ?@ ?@@?he@@ ?W20Y? ?@ ?@@?he@@ O.M? ?@ ?@@?he@@ W20Y ?@ ?@@?he@@ ?W.M ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@ W.Y? ?@ ?@@?he@@ ?W.Y ?@ ?@@?he@@L? W.Y? ?@ ?@@Lhe@V1?hf?W.Y ?@ ?@V1he@?@?hfW.Y? ?@ ?@?@he@?@?he?W.Y ?@ ?@?@he@?@?heW.Y? ?@ ?@?@he@?@?h?W.Y ?@ ?@?@he@?@?hW.Y? ?@ ?@?@he@?@?g?O.Y ?@ ?@?@he@?@?f?W20Y? ?@ ?@?@he@?@?fW.M? ?@ ?@?@he@?@?e?W.Y ?@ ?@?@he@?@?eW.Y? ?@ ?@?@he@?@??W.Y ?@ ?@?@he@?@?W.Y? @? ?@ ?@?@he@?@W.Y @? ?@ ?@?@he@?@(Y? @L ?@ ?@?@he@?@H ?J@1 ?@ ?@?@he@@@? ?7Y@ ?@ ?@?@h?J@?@? ?@?@ ?@ ?@?@hW&5?@? ?@?@ ?@ ?@?@g?W&@H?@? ?@?@ ?@ ?@?@gW.Y@e@? ?@?3L? ?@ ?@?@f?W.Y?@e@? ?@?N1? ?@ ?@?@fW.Y??@e@? ?@e@? ?@ ?@?@f7He?@e@? ?@e@? ?@ ?@?@e?J5?e?@e@? ?@e@? ?@ ?@?@eW.Y?e?@e@? J5e@? ?@ J5?@?W.Yf?@e@? 7He3L ?@ 7H?@W.Y?f?@e@? @?eN1 ?@ @??@(Yg?@e@? @?e?@ ?@ @?J@H?g?@e@? @?e?@ ?@ @?7@h?@e@? @?e?@ ?@ @W@@h?@e@? @?e?@ ?@ @(Y@h?@e@? @?e?3L? ?@ ?J@H?@h?@e@? @?e?N1? ?@ W&@??@h?@e@? @?f@? ?@ ?W.Y@??@h?@e@? @?f@? ?@ W.Y?@??@h?@e@? @?f@? ?@ ?W.Ye@??@h?@e@? @?f3L ?@ ?7H?e@??@h?@e@? @?fN1 ?@ J5f@??@h?@e@? ?J5?f?@ ?@ ?W.Yf@??@h?@e@? ?7H?f?@ ?@ W.Y?f@??@h?@e@? ?@g?@ ?@ @?f?W.Yg@??@h?@e@? ?@g?@ ?@ @?fW.Y?g@??@h?@e@? ?@g?3L? ?@ @?e?W.Yh@??@h?@e@? ?@g?N1? ?@ @?e?7H?h@??@h?@e@? ?@h@? ?@ @?eJ5he@??@hJ5e@? ?@h@? ?@ ?J@L?W.Yhe@??@h7He@? ?@h@? ?@ ?7R)T.Y?he@??@h@?e3L ?@h@? ?@ ?@?@(Yhf@??@h@?eN1 ?@h3L ?@ ?@?@H?hf@??@h@?e?@ ?@hN1 ?@ ?@@@hf?J5??@h@?e?@ J5h?@ ?@ J@?@hf?7H??@h@?e?@ 7Hh?3L? ?@ 7@?@hf?@e?@h@?e?@ @?h?N1? ?@ ?@?@ ?@ ?W.Y@??N1?he?@e?N1?f?J5?e?N1?J@1? W.Y?@?e@?he?@f3Lf?7H?f@?7R'L ?J@5?@hf?@e?@h@?e?@ W&@H?3L?he?@e?3L?g@?e?3L??)X? ?J5?he?3L??@L?hf O.Y?he?N1??@1?hf @?he3Le?@hg @?heN1e?@hg ?@ ?@?@ ?@ ?W.Ye@?e@?heJ5fN1f?@g3T5?N1 ?7U??J5?e3LfW-X?e7Hf?3=?eC5gV+Y??3L?hfO.M? J@)X?7H?eN1?)X?7R1?e@?f?V46T20Yhe?V/XheO20Y 7@V/X@f?3T@)T5?3T-X@?g?I+M V/K?fO2@@0M ?W2@0Y 3L?@@?hf N1?@@Lhf ?3X@V1hf ?V4@?3L?he ?@ @5?V4@f?V+MI+Y?V+R4@? ?V4@@@@@0M ?N1?he ?@ ?O2@@@0Y 3Lhe ?@hO2@@@@@@@@@0M?@? ?@@@@@@@@@0Mhe?J5? ?@ ?7H? N)X?h?@)KhI4@@@@@?e ?@ J5 ?@ ?W.Y ?@ ?7H? ?@ J5 ?@ ?W.Y ?@ W.Y? ?@ 7H ?@ ?J5? ?@ O.Y? ?@@@@@@@@@@@@@@@0Y ?@?@?@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@? ?@@@@@@?hg ?@e@?@@@@@?W26Xf ?@e@?@?@?@?7YV1f ?@e@?@?@?@?@@@@f ?@e@?@?@?@?3Xg ?@e@?@?@?@?V4@?f 25
A summary of the characteristics of MonoBeads is shown in Table 2. Table 2.Characteristics of MonoBeads. Mono S strong cation exchange Charged group -O-CH2-CHOH-CH2-O- -O-CH2-CHOH-CH2-O -CHz-CHOH-CHz-N*(CHaJ3 -CHz-CHOH-CHzSO Topacity(e) 270-370 140-180 00) 2 ND ND c-lactalbumin (MW14 300) ND 1gG(MW150000) ND 75 Ribonudease(MW13700) N.D 75 Typical protein() 90-100 90-100 Typical enzyme aclivity rc 80 (um 3-11 212 2.12 short term 2.14 2-14 hestablevererod fm ieoiemnqohepHnrafoeegc 26
A summary of the characteristics of MonoBeads is shown in Table 2. Table 2. Characteristics of MonoBeads. Properties Mono Q Mono S Type of gel strong anion exchanger strong cation exchanger Charged group -O-CH2-CHOH-CH2-O- -O-CH2-CHOH-CH2-O- -CH2-CHOH-CH2-N+(CH3)3 -CH2-CHOH-CH2SO3 - Total ionic capacity (µmoles/ml gel) 270-370 140-180 Total protein binding capacity (mg/ml gel) Thyroglobulin (MW 669 000) 25 N.D. HSA (MW 68 000) 65 N.D. a-lactalbumin (MW 14 300) 80 N.D. IgG (MW 150 000) N.D. 75 Ribonuclease (MW 13 700) N.D. 75 Typical protein recoveries (%) 90-100 90-100 Typical enzyme activity recoveries (%) >80 >80 Average particle size (µm) 10 ±0.5 10 ±0.5 MW range (proteins) up to 107 up to 107 working pH range* 3-11 3-11 pH stability** long term 2-12 2-12 short term 2-14 2-14 N.D. = Not determined Solvent restrictions: The ion exchangers are stable in alcohol/water solutions (C1-C4). 100% dimethyl sulphoxide, dimethylformamide, and formic acid can change the separation properties of the gel. Avoid oxidizing and reactive reagents. Detergents can be used if they are non-ionic or have the same charge as the gel. * working pH range refers to the pH range over which the ion exchange groups remain charged and maintain consistently high capacity. ** pH stability, long term refers to the pH interval where the gel is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures. 26
Flow rate The rigid monodisperse nature of the media enables high flow rates to be used on cmnnddowanodomceosennn Capacity ore size of the matrix.the capacities for large proteins as well as for smaller polypeptides and peptides pica m Table 3.Chromatographic properties of pr-packed com of MonoBeads. Properties PC1.6/5 HR 5/5 HR 10/10 HR16/10 otcolum0o0 Column volume (ml) 01 820 100 300 axoehegiomm 1.6x505x5010x10016x10035x10060x100 R6oaemsem 0.01-0.400.5-2.0upt06upt010pto32upt094 r(Pa) 2 25000250002500025000 2500025000 520 5-2040 40 60-90 60-90
Flow rate The rigid monodisperse nature of the media enables high flow rates to be used on MonoBeads columns. Normal recommended flow rates for high resolution separations are in the range 150 to 600 cm/h for HR 5/5 columns. Higher flow rates can be used during column washing and regeneration. In addition, the absence of buffering capacity means that buffer exchange and re-equilibration can be executed quickly and with small amounts of buffer. Details of the recommended flow rates to be used on the different columns are given in Table 3. Capacity The high substitution levels coupled with the large pore size of the matrix, the exclusion limit for globular proteins is 107 , give MonoBeads exchangers high capacities for large proteins as well as for smaller polypeptides and peptides. Typical saturation capacities are in the range of 60 mg protein per ml of gel and typical sample loading capacities are in the region of 25 mg of protein per ml of gel. Data on the saturation capacities for some specific proteins are given in Table 2. Table 3. Chromatographic properties of pre-packed columns of MonoBeads. Properties PC 1.6/5 HR 5/5 HR 10/10 HR 16/10 BioPilot Column 35/100 60/100 Column volume (ml) 0.1 1 8 20 100 300 Column dimensions 1.6x50 5x50 10x100 16x100 35x100 60x100 i.d. x bed height (mm) Recommended working flow rate range (ml/min) 0.01-0.40 0.5-2.0 up to 6 up to 10 up to 32 up to 94 Max operating 5 5 4 3 2 2 pressure (MPa) Number of theoretical plates per meter (N/m) 25 000 25 000 25 000 25 000 25 000 25 000 Normal separation times (min) 5-20 5-20 40 40 60-90 60-90 27
The titration curves for Mono Qand MonoS(Fig.10)show no buffering capaci- mg of the ge that the loading capacity does not vary with pH over the working neengrn1Kc 9 9 8 、 3 aiad Recovery fic int tions to the monobeads matrix are y ly recover ies are high.Recov of protein mass are typi cally 90-100%and of Table4.Protein activity recoveries (%)from MonoBeads columns Protein Mono Q MonoS N.D. Creatine Kinase A&ahle。Dehydrogenase 82 N.D.Not determined 28
The titration curves for Mono Q and Mono S (Fig. 10) show no buffering capacity which means that the loading capacity does not vary with pH over the working range of the gel. Fig. 10. Titration curves for Mono Q and Mono S. (Work by Pharmacia Biotech, Uppsala, Sweden.) Recovery Non-specific interactions to the MonoBeads matrix are very low and consequently recoveries are high. Recoveries of protein mass are typically 90-100% and of protein activity greater than 80%. Examples of protein activity recoveries are shown in Table 4. Table 4. Protein activity recoveries (%) from MonoBeads columns. Protein Mono Q Mono S b-Glucuronidase 106 N.D. b-Glucosidase N.D. 93 Phosphodiesterase 80 N.D. Creatine Kinase 90 N.D. Enolase N.D. 95 Lactate Dehydrogenase N.D. 102 Aldolase N.D. 94 N.D. = Not determined 28 ?@@?e@@ ?W2@@6X? @@e?@@? 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