Columns for HPLC. Columns for HPLC. Basic principles of preparative HPLC

Transcription

1 Basic principles of preparative HPLC The efficiency of HPLC for separation of one or several components from a complex mixture makes HPLC an important technique for preparative purifications. The difference between analytical and preparative HPLC concerns the aim of the separation. In analytical HPLC the aim is to separate all individual components of a mixture as completely as possible with subsequent identification of the peaks. In general, sample sizes are small. For 4 mm ID, typical sample sizes are µg analyte per g adsorbent in normal phase columns and µg analyte per g adsorbent in RP columns. For columns with smaller inner diameters correspondingly smaller samples are applied. Thus analytical HPLC often requires maximum separation efficiency of a column. Due to the small inner diameter, expenses for solvents and packings are low, with the result that in analytical HPLC costs for separation time (= solvent consumption) and packing material can be almost neglected for method development. n the contrary, in preparative HPLC development of a separation often involves detailed economical-chemical optimisation calculations. Due to the column dimensions, costs for solvents and packings or prepacked columns become more and more important with increasing column diameters. The aim of HPLC now is isolation of the desired product with defined purity, in maximum amounts and with minimum time. The important parameter is called production rate or throughput. This chapter discusses the most important parameters, which have to be taken into account in preparative method development. Definition of the production rate includes information about the required purity of the isolated product. That is, the user has to define the minimum resolution of the product peak from neighbouring peaks. The parameters determining the resolution between two peaks are relative retention α (ratio of both k values), the plate number of the column and the k value (formulas see chapter Basic terms and definitions on page 170). The relative retention is determined by the chromatographic system, i.e. mobile and stationary phases. Proper choice of column and eluent will produce the required selectivity and resolution of the chromatographic system for the sample to be separated. In practice, one often has to use a non-optimal system, e.g. because of sample solubility or because of incompatibility of a chromatographically desirable eluent with the following work-up or application steps of the isolated product after chromatography. For a detailed discussion of the term resolution please refer to the paper by L.R. Snyder 1). For maximising throughput, preparative HPLC columns are often overloaded (for details concerning overload phenomena see below). Under these conditions the resolution depends on the sample mass and on the injection volume. With increasing feed volume V 0 the resolution remains almost constant, until from a certain value it decreases linearly with further increase of the feed volume (for details about volume overload see below). Typical relation between feed volume and resolution w w min 1 R R max 1 w w min R = resolution R max = maximum resolution V 0 = peak width = minimum peak width = feed volume V 0 V 0 61

2 Basic principles of preparative HPLC When speaking about the production rate of a preparative separation, the term loadability of the column should be considered, too. According to general understanding, this is the maximum sample size (with defined sample mass and volume) under which a column still provides optimum selectivity. The chemical literature contains several propositions for definition of the loadability ) 6). In practice, however, these theoretical approaches are seldom used, because the maximum injection size is determined empirically: one injects increasing amounts, until the peaks just touch. However, it should be noted, that for the same injection size the loadability for dilute samples is higher than for concentrated ones 7). The parameters which are important for the optimisation of the mass loadability of a column can be described by the following formula. Mass loadability of a column M C 1 πr d P = lkda S C l M = maximum sample mass C 1, C = constants r = column radius l = column length K = partition coefficient d = packing density A S = adsorbent surface d P = particle diameter The significance of the individual parameters can be easily seen. However, two terms should be noted in detail: column length l and particle diameter d P. As can be seen from the term d P /l, the mass loadability of the column decreases with increasing plate number (l/d P is proportional to the plate number N). Experimentally, this relation can be easily shown with columns of different plate numbers. N The preceding diagram shows the dependence of the separation efficiency of two columns from the mass loading. The two columns were run under the same conditions, but with packings of different particle size (d P ) 8) If an increased loadability is required for a given separation efficiency, it is recommended to increase particle size and column length, the increase in column length being the square of the increase in particle diameter. This will produce a welcome side effect: the relative permeability of the column will also increase quadratically, and the pressure drop of the column will decrease quadratically as shown in the table below. Influence of particle size and column length on loadability and permeability for constant separation efficiency l [cm] d p [µm] rel. loadability rel. permeability As can be seen from the following formula, the volume loadability linearly depends on the dead volume of the column used, and otherwise it only depends on the k values of the components to be separated and on the separation efficiency of the column. Volume loadability of a column ) V L = V 0 ( α 1)k A ( + k A + k B ) N V 0 = dead volume V L = maximum overload volume α = relative retention (k B /k A ) N = plate number k A,k B = capacity factors column 1 column mass loading loadability column 1 loadability column g (component i) g (adsorbent) 6

3 Basic principles of preparative HPLC In addition to the loadability of the column used for a preparative separation another consideration can be important for the optimisation of the production rate 9). The production rate is directly proportional to the column diameter, the linear flow velocity of the mobile phase, the concentration of the component to be isolated (unless under mass overload conditions) and the term [1/N H 0 /l] 1/, where H 0 is the plate height of the column under ideal conditions, l is the column length, and N is the plate number required for separation of the desired product with the purity required. The following limiting cases can be distinguished: l < H 0 N: Here a negative value is generated under the square root with the result of a physically meaningless production rate: the plate number of the column is smaller than required. l = H 0 N: The column plate number is just sufficient for the separation. The corresponding column length is a critical value where the production rate is zero. l > H 0 N: Further increase of the column length results in increasing production rates. Production rate [mg/h] ,5 particle size [µm] 0 0 0,5 1 1,5 4 5 column length [m] Typical cases, which can cause column overload conditions, are e.g. samples with low solubility in the mobile phase or injection of too large a sample volume, highly concentrated samples, samples which are dissolved in a solvent with much better solvation characteristics than the mobile phase. Here sample concentration at the column inlet can cause problems. When considering overload phenomena, one has to distinguish between concentration overload, volume overload and mass overload conditions. Concentration overload The concentration of the analytes in the injected sample solutions is increased, while the injection volume is kept constant. With increasing overload peaks are more and more distorted, with the peak shape approaching a triangle. Fronting as well as tailing can occur. Simultaneously, with increasing overload the peak maxima are shifted, in most cases towards the dead time. This type of overload is called concentration overload. A concentration overload is only possible, if the solubility of the analytes in the sample solution is large enough. Volume overload If the solubility of the analytes is low, overload can only be obtained if, for a given sample concentration, the injection volume is continuously increased. Increasing sample volume results in peak broadening, approaching a rectangular shape. However, peaks remain symmetrical and from a certain overload peak heights remain constant. This type of overload is called volume overload. A characteristic of volume overload is constant retention volumes of the peak fronts even under overload conditions ). The rectangular peak shape obtained by volume overload is shown in the following chromatograms. The relation between column length and production rate can be seen in the diagram above, where typical values for production rates are plotted versus column length. The figure shows different curves for columns with different particle sizes and hence different separation efficiencies. It should be noted, that after a steep rise at relatively short column lengths the slope of these curves decreases drastically approaching a saturation value. This is reached when 1/N is much larger than H 0 /l. Since in practice optimisation of the production rate of a desired substance often results in overload conditions of the column, we briefly wish to discuss the related phenomena. 6

4 Basic principles of preparative HPLC Volume overload is demonstrated for the separation of a - component mixture of benzene, naphthalene and anthracene and a -component mixture of naphthalene and anthracene. The masses of the solutes injected were kept constant in all cases. -component mixture -component mixture Transition from elution development towards frontal analysis caused by volume overload sample volume A 10 µl 500 µl A B C B C sample volume 1 µl sample volume ml ml sample volume 1 ml sample volume ml A B C A B C sample volume ml sample volume 4 ml sample volume 6 ml sample volume ml sample volume 6 ml B+C A B C The constant retention volumes of the peak fronts combined with the rectangular peak shapes, however, allow a peak assignment with the aid of frontal analysis even for an otherwise insufficient resolution. When injecting such large volumes, certain precautions have to be taken when employing injection procedures with valve and sample loop. Improper injection can result in the concentration profile of the sample taking the form of a Poisson curve which will make frontal analysis difficult or impossible ). For the decrease of separation efficiency under volume overload conditions one can make the following estimate: The plate heights will increase by about 0% when the sample volume is about 1.5% of the column void volume, and they will about double when the sample volume increases to about.5% 5). sample volume 16 ml A+B+C A A+B B+C C 64

5 Basic principles of preparative HPLC Mass overload If the injected sample mass (calculated from injection volume and concentration) exceeds a certain value, the local concentration of the sample in the column can be so large, that equilibration is no longer possible: this case is called mass overload. Mass overload is much more complex than volume overload and is based on three main effects ) : Dispersion effects. The sample is distributed along the column by the mobile phase until it contacts sufficient adsorbent to permit equilibrium with the stationary phase. This phenomenon results in band spreading similar to volume overload. Deactivation of the adsorbent. In addition to dispersion effects a massive sample charge will occupy a significant portion of the stationary phase resulting in a changed effective polarity of the mobile phase compared to the stationary phase. This can cause reduced retention which will also affect components of the sample with lower concentration. Non-linear adsorption isotherms, caused by large sample concentrations, result in peak tailing. Thus, mass overload can be recognised by changed retention volumes of the peak fronts of all components of a mixture to be separated. Peak broadening and tailing will mainly affect the sample components which are present in excess relative to the column load capacity (see chromatograms on the left). Mass overload for the separation of benzene, naphthalene and anthracene B N A The figure shows mass overload for the separation of benzene, naphthalene and anthracene. The amount of benzene injected is increased from 180 µg via 8.1 mg to 16.9 mg, while the amounts of naphthalene and anthracene are kept constant. If the aim of a preparative separation is to obtain as much of the pure compound per time unit as possible, overload of the column will in most cases be necessary. In practice combinations of concentration and volume overload are most often used. With diluted samples volume overload will occur more often, while concentrated sample solutions will show a tendency towards concentration and mass overload. ften both effects are present and peaks approach the shape of a trapezoid. Concentration overload is to be preferred, because it allows separation of larger sample amounts. References: 1) L.R. Snyder, J. Chromatogr. Sci. 10 (197) 00 and 69 ) A. Wehrli, U. Hermann and J. F. K. Huber, J. Chromatogr. 15 (1976) 59 ) R. P. W. Scott, P. Kucera, J. Chromatogr. 119 (1976) 467 4) L. R. Snyder, Anal. Chem. 9 (1967) 698 5) W. Beck, I. Halasz, Z. Anal. Chem. 91 (1978) 40 6) T. Roumeliotis, K. K. Unger, J. Chromatogr. 185 (1979) 445 7) J. J. De Stefano, H. C. Beachell, J. Chromatogr. Sci 10 (197) 654 8) A. W. J. De Jong, H. Poppe, J. C. Kraak, J. Chromatogr. 09 (1981) 4 9) K. P. Hupe, H. H. Lauer, J. Chromatogr. 0 (1981) 41 10) V. R. Meyer, Praxis der Hochleistungs-Flüssigchromatographie, tto Salle Verlag, Frankfurt, 7. Aufl., 199 B B N A N A 65

6 Scale-up from analytical to preparative HPLC amount of adsorbent sample volume amount of analyte flow rate } = f(id) under analytical conditions to reach a resolution as high as possible. The higher the selectivity for a given phase system, the more a column can be overloaded. For preparative separations under overload conditions stationary phases with larger mean particle sizes are usually sufficient. Prerequisite for a successful transfer from established analytical methods to the preparative scale are stationary phases with equal selectivity for both methods. However, heavy overload can also change the chromatographic selectivity relative to analytical conditions. Scale-up factor For separation of larger amounts of a substance there are two general approaches: linear scale-up of the analytical system overloading the column In the case of linear scale-up the column length is kept constant and the column cross section is increased proportionally to the sample mass. Eluent flow and sample volume are adapted correspondingly. As in analytical HPLC, stationary phases with small particle sizes are used. The separation efficiency of the prep column is more or less the same compared with the analytical column. Peaks remain sharp and symmetrical. The procedure of linear scale-up is not very economical, since large columns and large amounts of eluents are required, and the substance yield is relatively low. For example, more than 5 kg stationary phase and 50 l mobile phase are necessary to separate less than 1 g of a substance mixture in one run. For this reason linear scale-up alone is only used in exceptional cases, e.g. if for difficult separations the high separation efficiency is needed for the isolation of (in most cases small amounts of) substances with very high purity. A better way is overloading the column, since for a given yield less stationary phase and less eluent are required. This procedure allows injection and separation of amounts in the lower milligram range on an analytical HPLC column. For preparative separations on the gram or kilogram scale overloading is combined with a linear scale-up of the chromatographic system. However, overloading a column always means a considerable loss of separation efficiency. For this reason, overloading the chromatographic system must show sufficient resolution. Consequently, for optimising a preparative separation, the chromatographic selectivity has to be optimised Procedure for scale-up In general, an analytical chromatogram is used as a starting point for a preparative separation. It is necessary to find conditions, which separate the sample mixture isocratically with good resolution, since gradients are not recommended for preparative separations, because they require large efforts in every respect. If necessary, proper sample preparation may be required. The better the resolution in the analytical chromatogram, the larger the load on the preparative column can be. After optimisation of the separation on the analytical column the maximum sample amount for injection of a concentrated solution is determined empirically. In the simplest case overload is increased, until the peaks in the chromatogram of the separation just do not yet overlap, allowing isolation of the peaks and, after removal of the mobile phase, obtaining the pure substances with 100% yield. Undiluted solutions are not favourable. It is not recommended to dissolve the sample in a stronger solvent than the mobile phase. Since for preparative work in most cases larger volumes are injected, the solvent can severely interfere with the equilibrium in the column, even making reproducibility of the chromatographic system impossible. Solubility of the sample in the mobile phase has to be good, because otherwise the column may get plugged. 66

7 Scale-up from analytical to preparative HPLC The scale-up factor After optimisation of the analytical separation the preparative column is to be considered. Transfer from an analytical to a preparative separation is easiest, if both are packed with the same stationary phase (linear scale-up). A key for successful transfer of results gained for the analytical column to the preparative scale is the proper scale-up factor. If the analytical or the preparative factor is not listed in the table below, it can be calculated form the following formula: Now every parameter relevant for the separation has to be multiplied with the scale-up factor. This includes flow rate, sample volume, sample mass, and the IDs of the capillaries. It is important during scale-up to increase the inner diameters of the capillaries proportionally, since otherwise the back-pressure of the system can become to large. For our programme of capillary tubing for HPLC please see the chapter Accessories on p. 166) L p d p M p = M a L a d a M L d a p = sample mass = column length = column diameter analytical column preparative column Scale-up factors and parameters for typical column dimensions Column dimensions [mm] Particle sizes [µm] NUCLESIL 5, 7, 10 NUCLEDUR 5 4 x 50 8 x x x 50 1 x x x x 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 5, 7, 10, 10, 1, 16, 0, 0, 50 Linear scale-up factor Typical sample mass * [mg] Amount of packing / column [g ±0%] Typical flow rate [ml] *for RP material; the maximum amounts given here always depend on the separation problem and on the sample composition. In some cases even half of the amounts given can cause dramatic overload, in other cases the maximum amounts can still give acceptable separations. 67

8 columns for preparative HPLC As with our analytical columns, our preparative columns feature capillary connections with UNF inner threads 10- ( 1 / 16 ), thus meeting today s standard in HPLC technology. They consist of stainless steel and are packed with NUCLESIL or NUCLEDUR packings from our range of silicas for HPLC. All packed columns are individually tested and supplied with the corresponding test certificate. We offer three different types of preparative columns: Standard-Prep, VarioPrep and Ecoprep. Standard-Prep columns are available in lengths of 15 to 500 mm and inner diameters of 10 mm, 1 mm and 50.8 mm. Standard-Prep guard columns are 50 mm long and available with the same inner diameters. Thus for scale-up the user can choose from a range of about two orders of magnitude in column diameters from the analytical 4 mm ID columns up to the 50.8 mm ID prep columns. VarioPrep columns have been developed in order to allow compensation of the dead volume, which could result at the column inlet after some time of operation, without need for opening the column. This special column technology is available as columns with one adjustable end fitting at the column inlet and a fixed end fitting at the column end. n request, we can also supply columns with two adjustable end fittings, an option which may e.g. be useful for frequent use of backflushing techniques. VarioPrep columns are available with lengths of 15 and 50 mm and inner diameters of 10, 1, 40 and 80 mm. Vario- Prep guard columns are 50 mm long and available with inner diameters of 10, 1 and 40 mm. EcoPrep columns are available with lengths of 0, 70, 15 and 50 mm. The column head is an enlarged version of the analytical EC columns (s. p. 15). The inner diameters are intermediate sizes of 8 and 16 mm. These columns are especially suited for SMB systems. Shorter columns (individually tested) and guard columns are available on request. n request, our preparative columns can also be custom-packed with other types of NUCLESIL packings as well as with NUCLEDUR silicas. In addition, they can be made in lengths other than those indicated in the tabulated summary on pages 69 to 7. Preparative columns different types of column end fittings 1 Standard-Prep column with 10 mm ID Standard-Prep column with 1 mm ID VarioPrep column with 1 mm ID 4 EcoPrep column with 16 mm ID 1 4 EcoPrep columns 5 Standard-Prep column with ID (flanged end fitting) 68

9 Preparative columns packed with NUCLESIL rdering information Length 0 mm 70 mm 15 mm 50 mm Guard columns 50 mm Base deactivated RP phases NUCLESIL C 18 HD Particle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, monomeric coating, 0% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID VarioPrep columns 10 mm ID mm ID EcoPrep columns 8 mm ID NUCLESIL C 18 HD Particle size 7 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, monomeric coating, 0% C; eluent in column acetonitrile / water EcoPrep columns 16 mm ID NUCLESIL PRTECT I Particle size 5 ± 1.5 µm, pore size 100 Å; special RP phase, endcapped, monomeric coating, 11% C; eluent in column acetonitrile / water VarioPrep columns 10 mm ID EcoPrep columns 8 mm ID mm ID n request, all preparative columns are available with any NUCLESIL or NUCLEDUR packing. Each column is individually tested and supplied with test chromatogram and test conditions Standard-Prep columns 69

10 Preparative columns packed with NUCLESIL rdering information Length 0 mm 70 mm 15 mm 50 mm Guard columns 50 mm Standard octadecyl phases NUCLEDUR C 18 ec Particle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 17.5% C; eluent in column acetonitrile / water NEW VarioPrep columns 10 mm ID mm ID 7600 NUCLESIL C 18 Particle size 5 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water VarioPrep columns 10 mm ID mm ID NUCLESIL C 18 Particle size 7 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID mm ( ) ID VarioPrep columns 10 mm ID mm ID mm ID EcoPrep columns 8 mm ID mm ID NUCLESIL C 18 Particle size 10 ± 1.5 µm, pore size 100 Å; octadecyl phase, endcapped, 15% C; eluent in column acetonitrile / water EcoPrep columns 16 mm ID NUCLESIL 10-7 C 18 Particle size 7 ± 1.5 µm, pore size 10 Å; octadecyl phase, endcapped, 11% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID NUCLESIL 00-7 C 18 Particle size 7 ± 1.5 µm, pore size 00 Å; octadecyl phase, endcapped, 6.5% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID VarioPrep columns 10 mm ID mm ID n request, all preparative columns are available with any NUCLESIL or NUCLEDUR packing. 70

11 Preparative columns packed with NUCLESIL rdering information Length 0 mm 70 mm 15 mm 50 mm Guard columns 50 mm Standard octyl phases NUCLESIL C 8 Particle size 7 ± 1.5 µm, pore size 100 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID VarioPrep columns 10 mm ID mm ID EcoPrep columns 8 mm ID mm ID NUCLESIL 00-7 C 8 Particle size 7 ± 1.5 µm, pore size 00 Å; octyl phase, not endcapped, ~% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID VarioPrep columns 10 mm ID mm ID Standard butyl phases NUCLESIL 10-7 C 4 Particle size 7 ± 1.5 µm, pore size 10 Å; butyl phase, endcapped, eluent in column acetonitrile / water Standard-Prep columns 10 mm ID n request, all preparative columns are available with any NUCLESIL or NUCLEDUR packing. VarioPrep columns 71

12 Preparative columns packed with NUCLESIL rdering information Length 0 mm 70 mm 15 mm 50 mm Guard columns 50 mm NUCLESIL 00-7 C 4 Particle size 7 ± 1.5 µm, pore size 00 Å; butyl phase, endcapped, ~% C; eluent in column acetonitrile / water Standard-Prep columns 10 mm ID mm ID 7150 VarioPrep columns 10 mm ID mm ID Standard phenyl phases NUCLESIL C 6 H 5 Particle size 7 ± 1.5 µm, pore size 100 Å; phenyl phase, not endcapped, eluent in column acetonitrile / water Standard-Prep columns 10 mm ID VarioPrep columns 10 mm ID mm ID Unmodified silica NUCLESIL 50-7 Particle size 7 ± 1.5 µm, pore size 50 Å; unmodified, eluent in column n-heptane Standard-Prep columns 10 mm ID mm ID 7150 NUCLESIL Particle size 7 ± 1.5 µm, pore size 100 Å; unmodified, eluent in column n-heptane Standard-Prep columns 1 mm ID VarioPrep columns 10 mm ID mm ID EcoPrep columns 8 mm ID mm ID n request, all preparative columns are available with any NUCLESIL or NUCLEDUR packing. n request, all types of preparative columns can also be packed with other NUCLESIL or NUCLEDUR phases. n request, VarioPrep columns are also available with two adjustable end fittings, with 80 mm ID or with different lengths. For preparative HPLC columns for special separation problems please see the following chapter Columns for special applications Each column is individually tested and supplied with test chromatogram and test conditions 7

13 Columns for special applications Summary In this chapter you will find HPLC columns for environmental analysis (from page 75) enantiomer separations (from page 79) biochemical separations (from page 9) nucleic acids and nucleic acid constituents proteins and peptides food analysis (from page 116) mono-, oligo- and polysaccharides hop constituents In the following chapter Products for GPC you will find columns for gel permeation chromatography (from page 14) polymer standards for GPC (from page 11) Certain difficult separation problems can often be solved better and much more rapidly with chromatographic columns which have been specifically developed for the purpose. Environmental analysis The quantitative determination of inorganic anions requires ion exchange columns which have been optimised for this separation. We offer three different types of columns to meet the different demands of anion analysis. The analysis of polycyclic aromatic hydrocarbons (PAHs) gains increasing importance. For this application too, only special columns can guarantee the efficiency required for complex samples. Enantiomer separation It is well known, that the two isomers of a natural chiral compound can differ substantially in their pharmacological activity depending on their absolute configuration. ften only one of the antipodes is pharmacologically active, while the other may be inactive, or even toxic. For synthetic drugs, the same holds true, and therefore it is necessary to control the optical purity of a substance. This is why we offer a complete line of chiral HPLC columns with different selectivities. Biochemical separations Chromatographic techniques are increasingly applied for the separation and purification of biologically active molecules. These methods take advantage of the chemical, biological and physical properties of the molecules. ften very complex mixtures of substances may require a combination of several chromatographic methods to obtain a pure product. Depending on the sensitivity of the molecules to be separated the eluents have to be adapted to the natural environment of these molecules. This is especially important for the purification of proteins or the isolation of long-chain nucleic acids. For these substances ion exchange chromatography and gel filtration methods are preferred. Peptides, nucleotides, oligonucleotides, oligo- and monosaccharides and amino acids behave more like conventional organic substances. For this reason they can be purified under highly efficient chromatographic conditions without consideration of the natural environment of the molecules. Nevertheless, their biological properties are maintained. For some questions in protein analysis, such as the sequence analysis of a protein or the determination of the content of an individual protein in biological fluids, preservation of the three-dimensional structure of the protein is not necessary. In this case purification can be performed under denaturing conditions with the highest possible selectivity. The same is true for the purification of nucleic acids. Food analysis A major chapter covers the analysis of mono- and oligosaccharides as well as sugar alcohols and organic acids. In addition to special silica phases we offer mixed-mode columns based on PS/DVB. These columns allow very efficient separations due to the combination of different mechanisms (ion exclusion, ion exchange etc.). For the analysis of hop constituents our special columns allow rapid routine separations without the problems usually encountered with this application. We offer a large number of columns for biochemical and food applications, which can be used for a wide variety of separation problems. In addition to columns for analytical and semipreparative separations, we also supply columns and stationary phases for pilot plant and production scale purifications. Thus scale-up from laboratory to production processes is easily possible. The following table presents a summary of our programme and helps in the selection of the proper column. For a detailed description of the packings used in the different columns please see the following pages. The table on page 9 presents a concise selection guide for our columns for biochemical separations. For chromatograms, we refer to our catalogue LC Applications which is available on request or visit our interactive collection of applications on the internet: The columns in this chapter have been developed specifically for difficult separation problems. The stationary phases used in these columns are in most cases not available as bulk packings, but only as packed columns. All columns are tested with special test mixtures for the respective purpose, thus we can guarantee the separation efficiency for these columns. If your separations require a metal-free environment, please ask for our custom-packed PEEK columns. 7

14 Columns for special applications Summary Summary of HPLC columns for special separations Separation/mechanism recommended column specification of the phase Environmental analysis anion exchange chromatography of inorganic anions polycyclic aromatic hydrocarbons (PAHs) Enantiomer separation NUCLESIL 10 Anion NUCLESIL Anion II NUCLEGEL Anion I NUCLESIL C 18 PAH strongly basic silica-based anion exchanger strongly basic silica-based anion exchanger strongly basic polymer-based anion exchanger NUCLESIL 100 polymer-coated with C 18 groups ligand exchange NUCLESIL CHIRAL-1 covalently bonded amino acid Cu(II) complexes charge-transfer-, dipole-dipole interactions and others enantioselective binding to chiral protein surface structures formation of inclusion complexes Biochemical separations anion exchange chromatography of biomolecules cation exchange chromatography of biomolecules reversed phase chromatography of biomolecules reversed phase chromatography of small molecules gel filtration of biomolecules Food analysis NUCLESIL CHIRAL- NUCLESIL CHIRAL- RESLVSIL BSA-7 NUCLEDEX α-pm, β-pm, γ-pm and β-h NUCLEGEN DEAE NUCLESIL PEI NUCLEGEL SAX NUCLEGEL SCX NUCLESIL MPN NUCLESIL PPN NUCLEGEL RP NUCLEGEL RP 100 NUCLEGEL RP-C 18 NUCLESIL GFC NUCLEGEL GFC brush type phases based on silica protein phase (BSA) based on silica permethylated and underivatised cyclodextrins covalently bonded to silica DEAE anion exchanger based on silica polymeric covalently bonded polyethyleneimine network based on NUCLESIL 4000, weakly basic anion exchanger strongly basic polymer-based anion exchanger strong cation exchanger based on a macroporous polymer with sulphonic acid modification monomerically bonded alkyl chains on silica polymerically bonded alkyl chains on silica polystyrene divinylbenzene polymer small pore macroporous PS-DVB polymer C 18 modified polystyrene divinylbenzene polymer hydrophilic polyalcohol modification on silica macroporous polymer with hydrophilic surface mono- and oligosaccharides NUCLESIL Carbohydrate special amino phase based on silica sugars, alcohols, org. acids NUCLEGEL SUGAR Ca, Na, Pb NUCLEGEL SUGAR 810 H, Ca, Pb NUCLEGEL IN 00 A } PS-DVB resins with sulphonic acid modification in different ionic forms hop constituents NUCLESIL C 18 Hop silica with C 18 modification Gel permeation chromatography (GPC) water-insoluble substances NUCLEGEL GPC gel matrix polystyrene divinylbenzene water-soluble polymers NUCLEGEL aqua-h macroporous polymer with hydrophilic surface 74

15 HPLC columns for environmental analysis The determination of environmentally important substances in very low concentrations and very different matrices such as surface water, drinking water, soil or waste water requires application of columns which have been tailored for these special separations. Combined with sample preparation and sample enrichment (e.g. with our CHRMABND columns or CHRMAFIX cartridges for solid phase extraction, which are described in the chapter Solid phase extraction from page 188) such columns provide a high degree of separation efficiency and reproducibility. ur programme comprises HPLC columns for the determination of inorganic anions, and polycyclic hydrocarbons (PAH). Columns for the separation of polycyclic aromatic hydrocarbons (PAHs) Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment. A number of PAHs (e. g. benzo[a]- pyrene, -methylcholanthrene and benzanthracene have been proven to be carcinogenic. Thus control of the PAH content of food, water and soil is an important task for routine analysis. For choice and limiting values of the polycyclics we refer to the governmental regulations, which exist in may countries (e.g. EPA method 610 of the United States Environmental Protection Agency). PAH can be determined by different chromatographic techniques (TLC, GC, HPLC). Thus the 6 PAHs according to German drinking water specification (TV) can e.g. be analysed by TLC (see German Standard DIN 8 409), while a much larger number of polycyclic aromatics can be determined by GC or HPLC. For PAH analyses we have developed a specially modified C 18 phase, which allows efficient gradient separation of the 16 PAHs according to EPA. rdering information Length 50 mm 150 mm 50 mm Guard columns NUCLESIL C 18 PAH special octadecyl phase on silica for the separation of PAHs, polymeric coating, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column acetonitrile / water 70:0 ChromCart columns mm ID mm ID mm ID mm ID EC columns 1) mm ID mm ID mm ID mm ID PAH standard according to EPA for HPLC PAH standard for HPLC 16 PAHs according to EPA method 610 in acetonitrile (1 ml) for composition see chromatogram on page ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). 75

16 HPLC columns for environmental analysis Detection of the separated PAHs can be achieved by UV (50 to 80 nm), with diode array or with fluorescence detection at different wavelengths for excitation and emission. Acenaphthylene cannot be analysed with fluorescence detection. For cost-effective routine PAH analysis we recommend application of columns with mm ID, because this will reduce the eluent consumption to 0 40% with about doubled detection sensitivity compared to the 4 mm ID column. For rapid analysis we recommend a column length of 50 mm. This allows separation of the 16 PAHs according to EPA in just 9 minutes. References Determination of PASH in Diesel fuel by HPLC and photodiode-array detection; J. Bunot, W. Herbel, H. Steinhart, J. High Res. Chrom. 15 (199) GIT Spezial Chromat. (199) Rapid separation of 16 PAHs according to EPA Column: EC 50/4 NUCLESIL C 18 PAH, 50 x 4 mm ID, Cat. No Eluent A: Water Eluent B: Acetonitrile Gradient: from 55 to 100% B in.5 min; then.5 min at 100% B; finally in 0.1 min from 100 to 55% B Flow rate: 1 ml/min Pressure: 5 0 bar Temperature: 5 C Detection: UV, 60 nm Peaks: (sample volume 10 µl) 1. Naphthalene 5 6. Acenaphthylene. Acenaphthene Fluorene 8 5. Phenantrene 6. Anthracene Fluoranthene Pyrene 1 9. Benz[a]anthracene Chrysene Benzo[b]fluoranthene 1. Benzo[k]fluoranthen 1. Benzo[a]pyrene 14. Dibenz[ah]anthracene 15. Benzo[ghi]perylene 16. Indeno[1,,-cd]pyrene 0 10 min Separation of the PAH standard according to EPA (Cat. No. 79) Column: CC 150/4 NUCLESIL C 18 PAH, 150 x 4 mm ID, Cat. No Eluent A: Methanol water (80:0) Eluent B: Acetonitrile tetrahydrofuran (9:7) Gradient: 0 100% B in 10 min, then 5 min at 100% B 7 Flow rate: 1 ml/min 5 Pressure: 140 bar 6 Temperature: 0 C 9 Detection: UV, 60 nm Peaks: (10 µg/ml each in acetonitrile) 4 1. Naphthalene. Acenaphthylene 10. Acenaphthene Fluorene 5. Phenantrene 6. Anthracene 1 7. Fluoranthene 8. Pyrene 9. Benz[a]anthracene Chrysene 11. Benzo[b]fluoranthene 1. Benzo[k]fluoranthene Benzo[a]pyrene 14. Dibenz[ah]anthracene 15. Benzo[ghi]perylene 16. Indeno[1,,-cd]pyrene min 76

17 HPLC columns for environmental analysis Columns for the separation of inorganic anions Analytical monitoring of the environment with respect to inorganic compounds today is the domain of ion chromatography. ur columns NUCLESIL 10 Anion and Anion II contain special silica-based anion exchangers. They are stable in the ph range 7.5. For applications which, due to the sample composition, require ph values outside this range, we offer the polymer-based column NUCLEGEL Anion I. A special feature of this column is that it can also be used for the analysis of fluoride ions. rdering information Length 10 mm 50 mm Guard columns NUCLESIL 10 Anion strongly basic silica-based anion exchanger, particle size 10 ± 1.5 µm, pore size 100 Å, exchange capacity 800 µval/g; eluent in column 5 mm salicylate buffer ph 4 EC columns 1) 4 mm ID NUCLESIL Anion II strongly basic silica-based anion exchanger, particle size 10 ± 1.5 µm, pore size 00 Å, exchange capacity 50 µval/g; eluent in column mm potassium hydrogen phthalate buffer ph 5.6 ChromCart columns 4 mm ID EC columns 1) 4 mm ID NUCLEGEL Anion I strongly basic polymer-based anion exchanger, particle size 10 µm; eluent in column 4 mm salicylate buffer ph 7.8 Valco type columns ) 4.6 mm ID All columns and ChromCart guard column cartridges are supplied in packs of 1. 1) As guard column for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). ) Valco type guard column cartridges are 1 x 4 mm, require guard column holder C (Cat. No ) and are supplied in packs of. 77

18 HPLC columns for environmental analysis NUCLESIL Anion columns based on silica ur columns EC 50/4 NUCLESIL 10 Anion and EC 50/4 NUCLESIL Anion ll and the corresponding Chrom- Cart columns are packed with spherical silica packings modified with quaternary ammonium groups, i.e. strongly basic anion exchangers; however, they differ considerably in their exchange capacity and in the pore size of the NUCLE- SIL silica. The exchange capacity of the NUCLESIL 10 Anion packing is approximately 800 µval/g, that of the NUCLESIL Anion II packing is about 50 µval/g. For this reason the columns should be operated with buffers of different concentrations. The recommended values are 5 mmol/l phthalate for NUCLESIL 10 Anion and mmol/l for NUCLESIL Anion II, respectively. Another consequence is, that both columns require different detection methods and feature different detection limits. NUCLESIL Anion II columns are mainly operated with conductivity detection or negative UV detection and allow determination in the ppm range. The column NUCLESIL 10 Anion is mainly used with RI detection resulting in a 10fold lower sensitivity. n the other hand due to the lower capacity columns NUCLESIL Anion II require the injection or rather dilute samples, i.e. clearly below 50 ppm per component (see upper figure). As eluent we recommend phthalate buffers with weakly acidic ph, although other buffer systems might be used as well. In this context we want to mention the corrosive properties of salicylate buffers. Salicylate anions react with iron ions, which may originate from the stainless steel of the instruments used (pumps, capillary tubing etc.) to produce a coloured complex which will interfere with UV detection. NUCLEGEL Anion polymer-based columns It should be noted that it is not possible to determine all inorganic anions with the silica-based columns. ne exception is fluoride which can only be separated at alkaline ph values because in an acidic medium it is irreversibly bonded to the silica. For this purpose we recommend our column NUCLEGEL Anion I, which is very well suited for this separation (see lower figure). It is packed with a macroporous, polymerbased anion exchanger. The base material is stable in the ph range from 0 to 14. For this reason any aqueous eluent can be used. Phthalate and salicylate buffers can also be applied. Using negative UV detection, less than 10 ppm can be analysed without enrichment of the samples. Separation of an anion standards Column: CC 50/4 NUCLESIL Anion II 50 x 4 mm ID, Cat. No Eluent: mm potassium hydrogen phthalate, ph 5.7 Flow rate: ml/min Detection: UV, 80 nm 1 Peaks: 1. H P 4. Cl. N 4 4. N 5. S Separation of inorganic anions min Column: VA 10/4.6 NUCLEGEL Anion I, 10 x 4,6 mm ID, Cat. No Eluent: 4 mm salicylic acid / Tris ph 7.8 Flow rate: 1 ml/min Detection: UV, 54 nm Peaks: F. Cl. N 1 4. Br 5. N 6. P 4 7. S For an increased life-time of all above-mentioned columns, we recommend the use of corresponding guard columns min

19 HPLC columns for enantiomer separation It is well known that the two isomers of a natural chiral compound can differ substantially in their pharmacological activity depending on their absolute configuration. ften only one of the antipodes is pharmacologically active, while the other may be inactive, or even toxic. For synthetic drugs, the same holds true, and therefore it is necessary to control the optical purity of a substance. NUCLEDEX columns enantiomer separation based on cyclodextrins, covalently bonded to NUCLESIL silica ur cyclodextrin-based HPLC columns NUCLEDEX β- H, α-pm, β-pm and γ-pm are very well suited for the separation of racemates, constitutional and configurational isomers under reversed phase conditions. A large number of racemates, including pesticides and pharmaceuticals, have been separated using these phases which are based on our well-known spherical silica NUCLESIL. Length 00 mm Guard columns NUCLEDEX screening kit (Cat. No. 7190) consists of one CC 0/4 each with NUCLEDEX β-h, α-pm, β-pm and γ-pm and a ChromCart column holder 0 mm NUCLEDEX β-h β-cyclodextrin chemically bonded to NUCLESIL silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH H / 0.1% TEAE ph 4 (55:45) ChromCart columns 4 mm ID EC columns 1) 4 mm ID NUCLEDEX α-pm permethylated α-cyclodextrin chemically bonded to NUCLESIL silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH H / 50 mm phosphate ph (70:0) ChromCart columns 4 mm ID EC columns 1) 4 mm ID NUCLEDEX β-pm permethylated β-cyclodextrin chemically bonded to NUCLESIL silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH H / 0.1% TEAE ph 4 (65:5) ChromCart columns 4 mm ID EC columns 1) 4 mm ID NUCLEDEX γ-pm permethylated γ-cyclodextrin chemically bonded to NUCLESIL silica, particle size 5 ± 1.5 µm, pore size 100 Å, eluent in column CH H / 0.1% TEAE ph 4 (55:45) ChromCart columns 4 mm ID EC columns 1) 4 mm ID All columns and guard column cartridges (8 mm) in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). 79

20 HPLC columns for enantiomer separation Separation mechanism The chiral selectors of the NUCLEDEX phases are cyclodextrins, which are covalently bonded to the silica matrix via a spacer, resulting in hydrolytically stable adsorbents The mean diameter of the spherical silica particles is 5 µm with a pore size of 100 Å. H H H H H H H H H H Structure of β-cyclodextrin Cyclodextrins are cyclic oligosaccharides consisting of glucose units. They are formed by degradation of starch by bacillus macerans or bacillus circulans under action of cyclodextringlycosyltransferase. As a first approximation, the cyclic structure of a cyclodextrin ring can be described as a hollow truncated cone. β-cyclodextrin contains seven glucose units, the opening of the cavity is about 7.5 Å. α-cyclodextrin contains only six glucose units resulting in a smaller opening of the cavity. γ-cyclodextrin with its 8 glucose units is the largest cyclodextrin. The inner surface of the cavity is lipophilic, thus a nonpolar part of the sample molecule (e.g. phenyl or naphthyl substituents of suitable size) can penetrate into the cyclodextrin ring. This forms so-called inclusion complexes, the stability of which is responsible for the retention of a compound. The chiral sugar units of the cyclodextrins allow enantioselective interactions and thus racemate separations. Application The column NUCLEDEX β-h contains β-cyclodextrin with free hydroxy groups as chiral selector. Thus hydrogen bonds and dipole interactions between functional groups of the analyte and the primary hydroxyl groups (small opening of the cyclodextrin) and secondary hydroxyl groups (large opening of the cyclodextrin) can occur. The phases NUCLEDEX α-pm, NUCLEDEX β-pm and NUCLEDEX γ-pm are based on permethylated cyclodextrins. While the chiral selector of the adsorbent NUCLEDEX β- PM is permethylated β-cyclodextrin, we use the smaller permethylated α-cyclodextrin for the phase NUCLEDEX α- PM. Correspondingly, the chiral selector for NUCLEDEX γ-pm is permethylated γ-cyclodextrin. For these phases all H H H H H H H H H H H hydroxy functionalities are replaced by methoxy groups, resulting in phase with different selectivity characteristics compared to NUCLEDEX β-h. Especially noticeable are the shorter retention times on NUCLEDEX β-pm. Permethylation of the cyclodextrin ring influences the number and type of possible interactions. The cage is enlarged and the hydrophobicity of the openings is increased. The ability of NUCLEDEX α-pm, β-pm and γ-pm to form hydrogen bonds is considerably lower. As shown in the figure below, the permethylated phases possess only proton acceptor properties due to the free electron pairs on the oxygen. N H H H proton acceptor and proton donor N H CH CH only proton acceptor Enantiomer separation of dichlorprop Column: CC 00/4 NUCLEDEX α-pm Cat. No Sample volume: 1 µl Eluent: Methanol / 50 mm NaH P 4 ph,0 (65:5, v/v) Flow rate: 0,7 ml/min Pressure: 170 bar Temperature: 0 C Detection: UV, 0 nm Cl Cl CH C H CH 0 10 min 80

21 HPLC columns for enantiomer separation For the example of the barbiturate mephobarbital, also known as prominal, methylation of the cyclodextrin clearly results in a better and more rapid separation of the antipodes. In NUCLEDEX β-h, the free hydroxy groups of the oligosaccharide result in unnecessary interaction, which causes longer retention times without additional chiral discrimination. n the one hand, NUCLEDEX β-pm shows good selectivity for some compounds which cannot be separated on β- H, e.g. the pesticide derivatives mecoprop methyl and dichlorprop methyl. n the other hand, compounds like chlorthalidone require the free hydroxy groups for enantioselective interactions. Such compounds can only be separated on the phase NUCLEDEX β-h. NUCLEDEX α-pm allows separation of the herbicides mecoprop and dichlorprop as free carboxylic acids. In addition, this phase shows good selectivity for aromatic oxiranes such as trans-stilbene oxide or styrene oxide. Due to their large molecules, steroids can best be separated on permethylated γ-cyclodextrin, i.e. on NUCLEDEX γ- PM. These few examples show that the four phases ideally complement each other with respect to selectivity. For numerous separations of the NUCLEDEX phases please ask for our catalogue LC Applications or on our website: Enantiomer separation of mephobarbital (prominal) Column: EC 00/4 NUCLEDEX β-pm; 00 x 4 mm ID; Cat. No Eluent: Methanol / 0.1% TEAA ph 4.0 (55 : 45, v/v) Flow rate: 0.7 ml/min Pressure: 180 bar Detection: UV, 54 nm Sample: Prominal Inj.volume: 1 µl Column: Eluent: Flow rate: Detection: Separation of D,L-estrone on NUCLEDEX phases Column: Eluent: Flow rate: Detection: Enantiomer separation of styrene oxide EC 00/4 NUCLEDEX α-pm; 00 x 4 mm ID; Cat. No Methanol / 0.1% TEAA ph 4.0 (60 : 40, v/v) 0.7 ml/min UV, 0 nm a) CC 00/4 NUCLEDEX β-pm, Cat. No b) CC 00/4 NUCLEDEX γ-pm, Cat. No Acetonitrile/water (45:55, v/v) 0.8 ml/min UV, 80 nm 0 10 min /10600 CH CH a) b) H N N CH min min min For ordering information of NUCLEDEX columns please see page

22 HPLC columns for enantiomer separation Separation of chlorthalidone on NUCLEDEX β-h Eluent: methanol 0.1% TEAA (55:45), ph 4 Flow rate: 0,7 ml/min Detection: UV, 54 nm CC 0/4 CC 00/ As is well known, it is often difficult to predict the chances for separation of an unknown enantiomeric mixture on a chiral phase. In most cases trial and error is the only way to find the proper column. For this case we offer our NUCLEDEX CC screening kit (Cat. No ) consisting of one ChromCart column CC 0/4 each with NUCLEDEX β-h, α-pm, β-pm, γ-pm and a ChromCart column holder 0 mm This kit is especially designed for method development of enantiomer separations for rapid and economical selection of proper chiral phases 0 min min 10 Separation of mecoprop on NUCLEDEX α-pm Eluent: methanol 50 mmol NaH P 4 (70:0), ph Flow rate: 0,7 ml/min Detection: UV, 0 nm CC 0/4 CC 00/ min min Separation of mecoprop methyl on NUCLEDEX β-pm Eluent: methanol 0.1% TEAA (65:5), ph 4 Flow rate: 0.7 ml/min Detection: UV, 0 nm CC 0/4 CC 00/ min min 8

23 HPLC columns for enantiomer separation Column: Eluent: Flow rate: Detection: Enantiomer separation of benzoin EC 00/4 NUCLEDEX γ-pm, 00 x 4 mm ID, Cat. No Methanol / 0.1% TEAA ph 4.0 (55:45, v/v) 0.7 ml/min UV, 0 nm H The NUCLEDEX phases are especially suited for the control of optical purity as is shown below for the detection of 1% D-dansyl-leucine besides 99% of the L-isomer. Enantiomer separation of dansyl-d,l-leucine Sample: left: racemate, right: 1% D- besides 99% L-isomer L Column: EC 00/4 NUCLEDEX β-h, 00 x 4 mm ID, Cat. No Eluent: MeH 1% TEAA, ph 4,0 (65:5, v/v) Flow rate: 0,7 ml/min Detection: UV, 54 nm D L NMe S NH min Enantiomer separation of ibuprofen Column: CC 00/4 NUCLEDEX β-pm, 00 x 4 mm ID, Cat. No Eluent: MeH / 0.1% TEAA, ph 4.0 (60:40, v/v) Flow rate: 0.7 ml/min Pressure: 180 bar Detection: UV, 0 nm CH CH min It should also be noted that NUCLEDEX columns can be used for semipreparative separations as is shown below for the resolution of the isomers of mecoprop methyl. Semipreparative separation of mecoprop methyl Column: CC 00/4 NUCLEDEX β-pm, 00 x 4 mm ID, Cat. No Eluent: MeH / 0.1% TEAA, ph 4.0 (60:40, v/v) Flow rate: 0.7 ml/min Detection: UV, 54 nm 1 mg D µg Cl CH CCH min min For ordering information of NUCLEDEX columns please see page 79. 8

24 HPLC columns for enantiomer separation NUCLEDEX phases can also be used for positional and cis-trans-isomers, as is demonstrated in the following examples. Separation of cis-trans-isomers of γ-tocotrienol A. M. Drotleff, W. Ternes, Z Lebensm Unters Forsch A 06 (1998) 9 1 Column: EC 00/4 NUCLEDEX β-pm, 00 x 4 mm ID Cat. No Eluent: Acetonitril / water (58:4, v/v) Flow rate: 0.8 ml/min Detection: Fluorescence, mv λ em = 0 nm, λ ex = 95 nm Sample: cis,7 cis-γ-tocotrienol cis,7 trans-γ-tocotrienol trans,7 cis-γ-tocotrienol trans,7 trans-γ-tocotrienol NUCLEDEX β-pm is suited 50 for the separation of α-, β-, γ- and δ-tocotrienols. The elution sequence of the isomers was only determined for α-tocotrienol. Column: Eluent: Flow rate: Pressure: Detection: Peaks: 1. o-cresol. m-cresol. p-cresol Separation of o-, m- and p-cresol CC 00/4 NUCLEDEX β-h, 00 x 4 mm ID Cat. No Methanol / water 1 (50:50, v/v) 0.7 ml/min 170 bar UV, 54 nm H CH CH H C CH CH min min Separation of positional isomers of nitroaniline Column: EC 00/4 NUCLEDEX β-h, 00 x 4 mm ID, Cat. No Eluent: Methanol / 0.1% triethylammonium acetate ph 4.0, 50 : 50 (v/v) Flow rate: 0.7 ml/min Pressure: 180 bar Detection: UV, 54 nm Peaks (sample volume 1 µl): 1. m-nitroaniline. o-nitroaniline. p-nitroaniline N NH N NH N 1 NH min 84

25 HPLC columns for enantiomer separation Chromatographic conditions for column operation HPLC columns NUCLEDEX β-h, NUCLEDEX α-pm, NUCLEDEX β-pm and NUCLEDEX γ-pm are normally operated under reversed phase conditions. Methanol as well as acetonitrile in combination with water or buffer solutions are suited as eluents. The ph value of the eluent should be between and 8. The choice of the organic modifier influences the selectivity of the phases. Buffers should be either phosphate or triethylammonium acetate (TEAA). As shown below for the separation of dansyl-d,l-leucine, the separation efficiency can be improved by increasing the buffer concentration. The optimum column temperature should be between 0 and 50 C. With increasing temperature the retention time decreases. Lower temperatures in general increase the selectivity (see figure). The influence of the cyclodextrin size on the separation of enantiomer pairs of very similar compounds is shown below for the methyl, ethyl and propyl esters of the herbicide mecoprop. The adsorbent NUCLEDEX β-pm separates the three enantiomer pairs of the esters, but not the free carboxylic acid. The methyl ester already shows a low selectivity α. n the contrary, NUCLEDEX α-pm discriminates between the enantiomers of the esters with the same selectivity and also separates the free carboxylic acid mecoprop. Influence of the buffer concentration Column EC 00/4 NUCLEDEX β-h (Cat. No ); Sample dansyl-d,l-leucine; Eluent MeH/TEAA, ph 4.0, 65:5 (v/v); Flow rate 0.7 ml/min; Detection UV, 54 nm Influence of the ph value Column EC 00/4 NUCLEDEX β-h (Cat. No ); sample DNP-D,L-methionine; eluent MeH / 50 mm NaH P 4 (40:60 v/v); flow rate 0.7 ml/min; detection UV, 54 nm k () capacity factor 4 1 k (1) α ph value selectivity Influence of the temperature Column EC 00/4 NUCLEDEX β-h (Cat. No ); sample dansyl-d,l-threonine; eluent acetonitrile/50 mm NaH P 4, ph 6.5 (0:80v/v); flow rate 0.8 ml/min; detection UV, 54 nm capacity factor k (1) α k () Temperature [ C] selectivity 0.1% TEAA 1% TEAA Influence of the ester alkyl group on capacity factor and selectivity shown for mecoprop esters Columns EC 00/4 NUCLEDEX α-pm (Cat. No ) and EC 00/4 NUCLEDEX β-pm, (Cat. No ); sample: different mecoprop esters; eluent methanol/ 50 mm NaH P 4, ph.0 (70:0 v/v); flow rate 0.7 ml/min; detection UV, 0 nm min capacity factor 5 4 β-pm β-pm α-pm α-pm methyl ethyl n-propyl selectivity For ordering information of NUCLEDEX columns please see page

26 HPLC columns for enantiomer separation NUCLESIL CHIRAL columns NUCLESIL CHIRAL-1 enantiomer separation based on ligand exchange ur HPLC column NUCLESIL CHIRAL-1 has been developed for the separation of optically active compounds and control of optical purity based on ligand exchange chromatography. The stationary phase for this column contains as matrix our well-known, pressure-stable NUCLESIL 10, chemically bonded with L-hydroxyproline / Cu + complexes as chiral selectors. Separation mechanism Ligand exchange chromatography (LEC) for the separation of enantiomers was introduced in liquid chromatography by Davankov et al. 1) and extensively studied by Gübitz and coworkers ). A paper by Davankov describes the enantioselectivity of ligand exchange chromatographic systems ). In LEC the principal interaction mode between solute enantiomers and the chiral selector is the formation of ternary mixed-ligand complexes with a transition metal cation, in the case of NUCLESIL CHIRAL-1 Cu(II) 4). The asymmetric centres of the hydroxyproline result in the formation of diastereomeric complexes. Differences in the stability of the complexes cause chromatographic separation. Separation of amino acid enantiomers Column: EC 50/4 NUCLESIL CHIRAL-1 50 x 4 mm ID, Cat. No Sample: D,L-alanine D,L-threonine Eluent: 0.5 mm CuS mm CuS 4 Flow rate: 1 ml/min 0.8 ml/min Pressure: 60 bar 65 bar Temperature: 60 C 60 C Detection: UV, 50 nm UV, 40 nm min min 10 rdering information Length 50 mm Guard columns NUCLESIL CHIRAL-1 L-hydroxyproline/Cu + complexes chemically bonded to silica, particle size 5 ± 1.5 µm, pore size 10 Å; eluent in column 0.5 mm copper sulphate solution ChromCart columns 4 mm ID EC columns 1) 4 mm ID All columns and guard column cartridges (8 mm) in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). 86

27 HPLC columns for enantiomer separation Chromatographic conditions for column operation As mobile phases for NUCLESIL CHIRAL-1 we recommend CuS 4 -containing aqueous eluents. The optical resolution can be optimised by changing the mobile phase composition. Cu + concentration Higher Cu(II) concentrations shorten retention times of the analytes. The Cu(II) concentration should be between 0. and mmol/l. rganic modifiers Addition of organic solvents like acetonitrile or methanol decreases retention times and improves peak symmetry. Temperature Increased temperatures improve column efficiency and result in shorter retention times. We recommend a temperature of 60 C and a maximum pressure not exceeding 140 bar. Application of NUCLESIL CHIRAL-1 A successful separation of enantiomers can be expected if the sample molecule has two polar functional groups with the correct spacing, which can form chelate complexes with the copper ions. Therefore, this column is very suitable for separation of α-amino acids with their amino and carboxylate groups. α-hydroxycarboxylic acids (e.g. lactic acid), N-alkylα-amino acids and similar molecules are also candidates for this method. ptical isomers separated so far include D,L-alanine, D,Larginine, D,L-asparagine, D,L-citrulline, D,L-methionine, D,Lphenylalanine, D,L-phenylglycine, D,L-proline, D,L-threonine and D,L-valine. Enantiomer separation of an α-hydroxycarboxylic acid Column: EC 50/4 NUCLESIL CHIRAL-1 50 x 4 mm ID, Cat. No Eluent: 0.5 mm copper sulphate Flow rate: 0.8 ml/min Temperature: 80 C Detection: UV, 40 nm Sample: (±)-lactic acid Sample volume: 1 µl The enantiomeric resolution of N-methyl-α-amino acids, α- alkyl-α-amino acids and α-amino alcohols on NUCLESIL CHIRAL-1 columns is reported by H. Brückner et al. 5), 6). H. Skopan et al. 7) described the application of NUCLESIL CHIRAL-1 columns for the control of optical purity of -hydroxycarboxylates from enzymatic reactions. Thomas and Surber described the enantiomer analysis of a HIV antiinfective nucleoside 8). For examples of enantiomeric separations with NUCLE- SIL CHIRAL-1 columns please see our catalogue LC Applications. References 1) V.A. Davankov, S.V. Rogozhin, A.V. Semechkin and T.P. Sachkova, J. Chromatogr. 8 (197) 59 V.A. Davankov, Adv. Chromatogr. 18 (1980) 19 ) Separation of the optical isomers of amino acids by ligand exchange chromatography using chemically bonded chiral phases G. Gübitz, W. Jellenz and W. Santi, J. Chromatogr. 0 (1981) ) Enantioselectivity of complex formation in ligand-exchange chromatographic systems with chiral stationary and/or mobile phases V.A. Davankov, A.A. Kurganov and T.M. Ponomarova, J. Chromatogr. 45 (1988) ) Applications and limitations of commercially available chiral stationary phases for high performance liquid chromatography R. Däppen, H. Arm and V. R. Meyer, J. Chromatogr. 7 (1986) 1 0 5) Enantiomeric resolution of N-methyl-α-amino acids and α-alkyl-α-amino acids by ligand exchange chromatography H. Brückner, Chromatographia 4 (1987) 75 6) Determination of α-alkyl-α-amino acids and α-amino alcohols by chiral phase capillary gas chromatography and reversed phase high performance liquid chromatography H. Brückner, I. Bosch, Th. Graser and P. Fürst, J. Chromatogr. 95 (1987) ) Ein Biokatalysator zur Herstellung von (R)- und (S)--Hydroxycarbonsäuren H. Skopan, H. Günther and H. Simon, Angew. Chem. 99 (1987) ) Preparative separation and analysis of the enantiomers of [ H] Abbott 6999, an HIV anti-infective nucleoside, by ligand exchange HPLC S. B. Thomas and B. W. Surber, J. Chromatogr. 586 (1991) min 87

28 HPLC columns for enantiomer separation NUCLESIL CHIRAL- and CHIRAL- for organic eluent systems The chiral columns NUCLESIL CHIRAL- and CHIRAL- have been developed for control of optical purity of compounds under normal phase conditions. Main components of the eluent systems are thus hydrocarbons besides polar organic modifiers. Length 50 mm Guard columns EC columns NUCLESIL CHIRAL- N-(,5-dinitrobenzoyl)-D-phenylglycine chemically bonded to silica, brush type phase, particle size 5 ± 1.5 µm; eluent in column n-heptane / isopropanol / trifluoroacetic acid 100:0.5:0.5 4 mm ID NUCLESIL CHIRAL- optical antipode of CHIRAL- chemically bonded to silica as above 4 mm ID As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). ChromCart guard column cartridges for NUCLESIL CHIRAL- and CHIRAL- are identical, measure 8 x 4 mm ID and are supplied in packs of, all other columns in packs of 1. Separation of the optical isomers of fenoprop methyl Column: EC 50/4 NUCLESIL CHIRAL-, 50 x 4 mm ID, Cat. No Sample: Fenoprop methyl Volume: µl Eluent: n-heptane / i-propanol / TFA (100:0.5:0.05, v/v) Flow rate: 1.0 ml/min Pressure: 80 bar Detection: UV, 0 nm Cl Cl Cl CH C CH CH NUCLESIL CHIRAL- Separation mechanism With the column NUCLESIL CHIRAL-, as chiral substance D-dinitrobenzoylphenylglycine is covalently bonded to the NUCLESIL silica matrix via a spacer. NUCLESIL CHIRAL- is a brush type phase. Chiral brush type phases for HPLC were first used by Mike s et al. 1) for the separation of racemic helicenes. Later, the groups of Pirkle and of i synthesised a large number of these phases. Although a lot of work has been done in this field, the separation mechanism is not completely understood in all instances. However, there is no doubt that charge-transfer interactions, hydrogen bonds, dipole-dipole interactions and steric effects are involved ). Chromatographic conditions for column operation The separation is performed using nonpolar organic mobile phases (n-heptane, isooctane) with polar organic additives such as tetrahydrofuran, alcohols, chlorinated hydrocarbons or similar. ften addition of a small amount of strong acids (e.g. trifluoroacetic acid) to the mobile phase will considerably improve separation of the isomers. The solubility of basic compounds can easily be enhanced by a simple derivatisation step (e.g. with benzoyl chloride or,5-dinitrobenzoyl chloride). ^ min 88

29 HPLC columns for enantiomer separation Application NUCLESIL CHIRAL- is recommended for the analysis of stereoisomers such as separation of enantiomers and diastereomers control of optical purity of plant protectives (pesticides, e.g. propionic acid derived herbicides ) ), pharmaceuticals etc. for product control in chiral organic syntheses For control of the optical purity of a substance, with the two columns NUCLESIL CHIRAL- and NUCLESIL CHIRAL- the chromatographer now has the option to select conditions such that the minor enantiomer, which is present as an impurity, is eluted before the main peak. Thus, overlapping peaks are avoided. This makes an exact quantification of the impurity much easier. Conditions for operation are identical for the columns NUCL- ESIL CHIRAL- and NUCLESIL CHIRAL-. NUCLESIL CHIRAL- This HPLC column differs from NUCLESIL CHIRAL- only in the configuration of the chiral selector. For NUCLESIL CHIRAL- L-dinitrobenzoylphenylglycine is used. The selectivity towards a racemic mixture as well as the chromatographic resolution of the respective enantiomer pairs correspond exactly to the column NUCLESIL CHIRAL-, however, the elution sequence of the enantiomers is reversed. Control of optical purity of mecoprop methyl on NUCLESIL CHIRAL- and CHIRAL- Column: a) EC 50/4 NUCLESIL CHIRAL-, Cat. No b) EC 50/4 NUCLESIL CHIRAL-, Cat. No Eluent: n-heptane / -propanol / trifluoroacetic acid (100 : 0.05 : 0.05, v/v) Flow rate: 1 ml/min Temperature: ambient Detection: UV, 0 nm Sample: Mecoprop methyl 90% ee Sample volume: 1 µl a) 95% D b) 95% D References 1) F. Mike s, G. Boshart and E. Gil-Av, J.Chromatogr. 1 (1976) 05 ) Chiral stationary phases for high performance liquid chromatographic separation of enantiomers. A minireview D.W. Armstrong, J.Liquid Chromatography 7 (S-) (1984) 5 76 ) Enantiomer resolution and assay of propionic acid-derived herbicides in formulations by using chiral liquid chromatography and achiral gas chromatography. M.D. Müller and H.-P. Bosshardt, J. Assoc. ff. Anal. Chem. 71 (1988) Column: Eluent: Flow rate: Detection: ^ Enantiomer separation of D,L-supidimide EC 50/4 NUCLESIL CHIRAL-, 50 x 4 mm ID, Cat. No Tetrahydrofuran / n-heptane (10:, v/v) 1.0 ml/min UV, 0 nm C N S N 5% L 5% L min 10 0 min min 4 89

30 HPLC columns for enantiomer separation RESLVSIL BSA-7 protein phase for separation of optical isomers based on silica and bovine serum albumin The chiral column RESLVSIL BSA-7 has been developed for the separation of optical isomers and demonstrated to be a highly successful tool for determination of the enantiomeric purity. The chiral stationary phase RESLVSIL BSA-7 is based on bovine serum albumin (BSA) covalently bonded to widepore silica. With respect to various chromatographic parameters this column can be characterised as follows: selectivity and resolution: speed: reproducibility: capacity: excellent very good excellent low to moderate The main advantages of the RESLVSIL column are extremely high selectivity easy regulation of retention by small changes in the mobile phase composition. This results in a high flexibility of the chromatographic system because an optical resolution may be optimised to fit given requirements. In combination with high-sensitivity detectors, very small amounts of a compound need to be injected on the column. Therefore, enantiomeric composition or purity can be determined very accurately in spite of the low capacity. Length 150 mm Guard column EC columns 1) RESLVSIL BSA-7 protein phase, bovine serum albumin (BSA) chemically bonded to NUCLESIL silica, particle size 7 ± 1.5 µm, pore size 00 Å; eluent in column 0.1 M phosphate buffer ph 7.5, % n-propanol 4 mm ID All columns and guard column cartridges (8 mm) in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). Separation mechanism The performance of the RESLVSIL column is based on selective interactions of proteins with low molecular compounds. Several proteins can undergo enantioselective interactions with a large number of pharmacologically active compounds. This effect was first used for chromatographic separations by Stewart and Doherty 1), who succeeded in resolving D- and L-tryptophan on bovine serum albumin (BSA) bound to agarose. Allenmark et al. ) made the method accessible to HPLC by binding BSA to HPLC-grade silica. Resolution of enantiomers on BSA which was irreversibly adsorbed to silica was studied by Erlandsson et al. ). The separation mechanism of protein columns is not known, although there is no doubt that it is based on principles of bioaffinity. It includes hydrophobic interactions (similar to a true reversed phase), interactions of polar groups and steric effects 4). Aubel et al. 5) investigated the effect of different pretreatments on the performance of BSA columns. Separation factors (α values) for selected representatives of various classes of compounds on RESLVSIL BSA-7 Class Compound α values on BSA-7 Aromatic amino acids D,L-kynurenin 7.5 DNP amino acids N-(,4-dinitrophenyl)-D,L-aspartic acid 7.7 Dansyl amino acids dansyl-d,l-glutamic acid. N-Aroyl amino acids N-(p-nitrobenzoyl)-D,L-alanine 17 Benzodiazepinones oxazepam 6.8 Sulphoxides o-carboxyphenyl-methyl sulphoxide.1 Coumarin derivatives warfarin 1.5 Lactams 4-amino--(p-chlorophenyl)butyric acid lactam 8 Aromatic hydroxy ketones benzoin

31 HPLC columns for enantiomer separation Applications Examples for the application of RESLVSIL are amino acid derivatives aromatic amino acids aromatic sulphoxide barbiturates benzodiazepinones benzoin and benzoin derivatives β-blockers coumarin derivatives RESLVSIL has proven useful for monitoring stereoselective microbial and enzymatic conversions 6), 8), 9). For examples of separations on the RESLVSIL column please see our catalogue LC Applications. Enantiomer separation of N-benzoyl-D,L-amino acids S. Allenmark 7) Column: EC 150/4 RESLVSIL BSA-7, 150 x 4 mm ID, Cat. No Eluent: 50 mm phosphate buffer ph % 1-propanol Flow rate: 0.70 ml/min Detection: UV, 5 nm Ia: Serine L L Ib: Alanine Ic: Phenylalanine Ia D Ib D L Ic D elution volume [ml] Chromatographic conditions for column operation Columns with protein stationary phases are demanding in that the chiral separation can be easily influenced by the chromatographic conditions such as ph, ionic strength, concentration of organic modifiers and temperature. Therefore, the optimum combination of these parameters should be determined for each separation problem. Mobile phase The RESLVSIL column is compatible with mobile phase systems consisting of aqueous buffers of ph between 5 and 8. Phosphate and borate buffers are adequate for this purpose. Avoid the use of ph extremes (< 5 or > 8). Retention and optical resolution can be regulated via ph, buffer strength ( M) and/or surface tension via small amounts of 1-propanol (0 5%) added as a co-solvent. Retention is always drastically reduced by as little as 1 to % 1-propanol and >5% is not recommended. ph and ionic strength will affect retention in a way not generally predictable. To some extent the columns respond like reversed phase columns, but please note that the columns will not tolerate mobile phase systems containing acetonitrile or methanol as used in reversed phase LC since the albumin will be denatured under such conditions. Upon a change of the mobile phase, please remember to allow sufficient time to elapse for complete re-equilibration. Generally, full reproducibility between runs will e obtained after a couple of hours. The mobile phase systems recommended for RESLVSIL offer the following advantages: Retention and optical resolution can be readily optimised by using several independent mobile phase parameters (ph, buffer strength, surface tension). The mobile phase is easily available, inexpensive, nontoxic and biocompatible. All types of high sensitivity detector systems can be used (UV, fluorescence, electrochemical etc.). Injection of aqueous samples can be made directly on to the column. The mechanically stable microparticles of the RESLVSIL column permit chromatography at high flow rates and pressures up to about 00 bar (400 psi). Load capacity For RESLVSIL BSA-7 optimum resolution is achieved with sample concentrations below 0. µmol per injection. Higher concentrations are likely to cause a decrease in resolution due to column overload. For biological samples you should use a reversed phase or RESLVSIL guard column or even a column switching technique to improve column life. If a higher column capacity is required we recommend using column BSA-7PX. 91

32 HPLC columns for enantiomer separation Column stability Chiral recognition of the RESLVSIL column is based on the interaction between covalently bonded bovine serum albumin (BSA) and low molecular compounds. Inherently, the protein structure is very susceptible to denaturing. For this reason RESLVSIL columns should be handled with care. For recommendation concerning the operation of these columns please see the chapter Mobile phase. When not in use, RESLVSIL columns should be stored in a refrigerator, equilibrated with azide-containing water. Separation of the optical isomers of omeprazole Column: EC 150/4 RESLVSIL BSA-7, 150 x 4 mm ID, Cat. No Sample: 15 µm omeprazole Volume: 0 µl Eluent: 0.05 M phosphate buffer ph % propanol-1 Flow rate: 1,0 ml/min Detection: UV, 50 nm H C CH CH N CH S N N H CH 0 4 min References 1) K.K. Stewart and R.F. Doherty, Proc. Natl. Acad. Sci. USA, 70 (197) 850 ) Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases III. ptical resolution of a series of N-aroyl D,L-amino acids by highperformance liquid chromatography on bovine serum albumin covalently bonded to silica. S. Allenmark, B. Bomgren and H. Borén, J. Chromatogr. 64 (198) 6 68 ) Direct analytical and preparative resolution of enantiomers using albumin adsorbed to silica as a stationary phase P. Erlandsson, L. Hansson and R. Isaksson, J. Chromatogr. 70 (1986) ) Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases IV. Molecular interaction forces and retention behaviour in chromatography on bovine serum albumin as a stationary phase S. Allenmark, B. Bomgren and H. Borén, J. Chromatogr. 16 (1984) ) Effects of pretreatment on the enantioselectivity of silicabound proteins used as high-performance liquid chromatographic stationary phases M.T. Aubel and L.B. Rogers, J. Chromatogr. 408 (1987) ) Some applications of chiral liquid affinity chromatography using bovine serum albumin as a stationary phase S. Allenmark, B. Bomgren and S. Andersson, Prep. Biochem. 14 (1984) ) ptical resolution of racemic compounds by means of HPLC on immobilized protein stationary phases - theory and application S. Allenmark, B. Bomgren and H. Borén, in "Affinity chromatography and biological recognition" (I. Chaiken, M. Wilchek, and I. Parikh. Eds.), Academic Press, New York, 198, S ) Enantioselective microbial degradation of racemates studied by chiral HPLC on silica-bound albumin. S. Allenmark, B. Bomgren, and H. Borén, Enzyme Microb. Technol. 8 (1986) ) Chiral reversed-phase liquid chromatographic monitoring of asymmetric carbonyl reduction by some yeast organisms S. Allenmark and S. Andersson, Enzyme Microb. Technol. 10 (1988) 177 For ordering information of RESLVSIL columns please see page 90 9

33 Summary of columns for bioanalysis and food analysis ph 8 NUCLESIL 15-5 GFC GFC ph 1 1 NUCLEGEL GFC 00-8 NUCLEGEL GFC ph 1 1 NUCLEGEL RP 00-5 NUCLEGEL RP Proteins RPC ph 8 NUCLESIL 00-5 C 4 MPN NUCLESIL 00-5 C 18 MPN ph 10 NUCLESIL C PPN NUCLESIL C 18 PPN IEC ph 8.5 NUCLESIL PEI ph 1 1 NUCLEGEL SAX NUCLEGEL SCX ph 8.5 NUCLESIL PEI IEC ph 1 1 NUCLEGEL SAX NUCLEGEL SCX Peptides NUCLESIL C 18 MPN ph 8 NUCLESIL 10- C 18 MPN NUCLESIL 00-5 C 4 MPN RPC NUCLESIL 00-5 C 18 MPN NUCLESIL C 18 PPN ph 1 1 NUCLEGEL RP * Amino acids also see HPLC columns for enantiomer separation from page 79 RPC ph 8 NUCLESIL C 18 MPN NUCLESIL 10- C 18 MPN Nucleosides ph 8 NUCLESIL C 18 MPN NUCLESIL 00-5 C 18 MPN RPC ph 10 NUCLESIL C 18 PPN NUCLESIL C 18 PPN Nucleotides, oligonucleotides DNA, RNA, restriction fragments, plasmids ph 1 1 NUCLEGEL RP * NUCLEGEL RP 00-5 ph 8.5 NUCLEGEN 60-7 DEAE NUCLESIL PEI IEC ph 1 1 NUCLEGEL SAX GFC ph 1 1 NUCLEGEL GFC 00-8 NUCLEGEL GFC NUCLEGEL GFC IEC ph 8 NUCLEGEN DEAE NUCLEGEN DEAE ph 1 1 NUCLEGEL SAX NUCLEBND AX* Polysaccharides GFC ph 1 1 NUCLEGEL GFC 00-8 NUCLEGEL GFC NUCLEGEL GFC Mono- and oligosaccharides Hop constituents ph 8 NUCLESIL Carbohydrate NUCLESIL NH ** NUCLEGEL IN 00 A NUCLEGEL SUGAR 810 H NUCLEGEL SUGAR Pb NUCLEGEL SUGAR 810 Pb ph 1 1 NUCLEGEL SUGAR Ca NUCLEGEL SUGAR 810 Ca NUCLEGEL SUGAR Na RPC ph 1 9 NUCLESIL C 18 Hop GFC = gel filtration chromatography IEC = ion exchange chromatography RPC = reversed phase chromatography * Please ask for our catalogue Bioanalysis ** see page 54 9

34 Ion exchange columns for biochemical applications NUCLEGEN columns for the separation of oligonucleotides and nucleic acids The NUCLEGEN family offers remarkable performance for biopolymers. It is a series of silica-based DEAE anion exchangers available with pore sizes of 60, 500 and 4000 Å, respectively. The choice of pore sizes and the chemistry of the surface coating was elaborated for present day problems in nucleic acid research. NUCLEGEN columns guarantee outstanding features with respect to resolution speed reproducibility purity life time recovery capacity solvent compatibility regeneration time The favourable interplay of these qualities is seen best from the separation of oligonucleotides, high molecular weight RNA and plasmid DNA. NUCLEGEN phases show neither swelling with salt or ph gradients nor a break-down of the column packing even with high flow rates. Reequilibration to starting conditions takes only a few minutes. NUCLEGEN columns are suited for every day use over a long period of time. It has been reported 1) that columns of 1.5 ml bed volume (4 mm ID x 15 mm) have been used with more than 100 l buffer without loss of resolution; this corresponds to about 1000 repetitive HPLC runs. For numerous separations of deoxyoligonucleotides, plasmids and DNA restriction fragments see our catalogue LC Applications or visit our website: Length 15 mm Guard columns 1) NUCLEGEN 60-7 DEAE DEAE anion exchanger based on silica, particle size 7 ± 1.5 µm, pore size 60 Å; eluent in column methanol EC analytical columns 4 mm ID Standard-Prep preparative columns 10 mm ID NUCLEGEN DEAE DEAE anion exchanger based on silica, particle size 7 ± 1.5 µm, pore size 500 Å, eluent in column methanol Valco type analytical columns 6 mm ID Standard-Prep preparative columns 10 mm ID mm ID, IWC NUCLEGEN DEAE DEAE anion exchanger based on silica, particle size 7 ± 1.5 µm, pore size 4000 Å, eluent in column methanol Valco type analytical columns 6 mm ID Standard-Prep preparative columns 10 mm ID mm ID, IWC IWC = columns inner wall coated with PTFE Columns are supplied in packs of 1. 1) ChromCart NUCLEGEN guard column cartridges are 0 mm long and supplied in packs of. They require the CC column holder 0 mm (Cat. No. 718). 94

35 Ion exchange columns for biochemical applications Capacity of NUCLEGEN DEAE columns Capacity [A 60 ] Phase column EC 15/4 VA 15/6 SP 15/10 NUCLEGEN 60-7 DEAE NUCLEGEN DEAE NUCLEGEN DEAE A 60 : Absorption unit at 60 nm; it corresponds to about 40 µg RNA or 50 µg DNA Good resolution is achieved if not more than 50% of the maximum binding capacity is loaded onto the column NUCLEGEN 60-7 DEAE for the separation of oligonucleotides NUCLEGEN 60-7 DEAE was particularly developed for the isolation of short nucleic acids. Synthetic oligonucleotides of defined length and sequence are required for modern genetic engineering and in molecular biology. The HPLC resins applied formerly were very limited with respect to resolution and recovery. Both disadvantages are overcome with our NUCLEGEN 60-7 DEAE; resolution is extended to chain lengths of 40 bases, and the recovery is > 95% 1), ). The packed columns are ready for use. Elevated temperatures for chromatography are not required. The high capacity of 00 A 60 /ml NUCLEGEN DEAE permits the use of small columns. For the isolation of oligonucleotides with chain lengths between and 50 the following buffer systems show a very good separation efficiency: Buffer A (low salt) 5 M urea 0.0 M K phosphate* ph % formamide, 0.0 M K phosphate* ph % methanol 0.0 M Na acetate ph % acetonitrile 0.0 M Na acetate ph Buffer B (high salt) 1 M KCl or LiCl, (NH 4 ) S 4 5 M urea, 0.0 M K phosphate*, ph M KCl or LiCl, (NH 4 ) S 4 50% formamide, 0.0 M K phosphate*, ph M KCl or LiCl, (NH 4 ) S 4 0% methanol 0.0 M Na acetate, ph M KCl or LiCl, (NH 4 ) S 4 0% acetonitrile 0.0 M Na acetate, ph * stock buffer 1 M K phosphate consists of 0.5 Mol KH P 4 (68.05 g) Mol K HP 4 (87.09 g) per litre The ph value is adjusted with H P 4 For preparative separations LiCl with the last-mentioned buffer system offers a great advantage, since nucleic acids may be directly precipitated from 4 M LiCl with ethanol/acetone without co-precipitation of the salt. The volume of the collected fractions and the LiCl concentration are determined (from the chromatogram) and the concentration is increased to 4 M LiCl in a vacuum centrifuge (e. g. Speed-Vac). To this concentrated sample five times its volume of ethanol/acetone (1:1) is added, precipitated 5 h at 0 C and centrifuged 1 h at 5000 rpm. Purification of full-length synthetic oligoribonucleotides K.R. Webster et al. BioTechniques 11 (1991) Column: EC 15/4 NUCLEGEN 60-7 DEAE Cat. No , equilibrated with 0 mm NaAc, 0% acetonitrile (ph 6.5) Gradient: 0 1,5 M KCl in 50 min Flow rate: ml/min Detection: UV, 60 nm Abs. 60 nm 0mer 1mer 18mer 4mer min [KCl] (M) 0 For ordering information of NUCLEGEN DEAE columns please see page

36 Ion exchange columns for biochemical applications Column: Buffer A: Buffer B: Gradient: A 60 0,0 0 Separation of oligo(ra) n EC 15/4 NUCLEGEN 60-7 DEAE 15 x 4 mm ID, Cat. No mm phosphate, ph 5.5, 5 M urea buffer A + 1 M KCl 0 100% B in 00 min Flow rate ml/min, 110 bar ambient temperature UV detection, 60 nm min Separation of a deoxyoligonucleotide 18mer H. Werntges, Diplomarbeit, University of Düsseldorf Sample: d(cgtcgtttaacaacgtcg) Column: EC 15/4 NUCLEGEN 60-7 DEAE 15 x 4 mm ID, Cat. No Buffer A: 40% acetonitrile, 0.0 M NaAc ph 5.5 Buffer B: buffer A M KCl Gradient: 0 50% B in 10 min % B in 50 min Flow rate: ml/min, 40 bar ambient temperature Detection: 60 nm As test mixture, oligo(ra) n, n = 0 is of advantage because it is easily prepared from poly(ra) with KH. For a test chromatogram 0.5 A 60 oligo(ra) n in 5 50 µl buffer A are injected onto the column and a linear gradient from 100% buffer A (0% B) to 100% buffer B (0% A) in 50 min is started at a flow rate of 1 ml/min. Under these conditions base line separation can be achieved up to n = 10, and up to n = 5 peaks are discernible. For resolution of higher chain lengths the gradient time should be increased from 50 min to 75 min or 100 min. With increasingly flatter gradients the resolution for higher chain lengths is improved. Better separations can be achieved with convex gradients, which allow for the smaller charge differences found with higher molecular weights. If the sample contains only long-chain oligonucleotides, it is timesaving to start the separation at higher ionic strength. Thus a separation of oligo(ra) 10 5 can be shortened from 50 min to 5 min by starting at 5% B (75% A). To prevent breakthrough of the sample, the ionic strength for starting the separation should be about 50 mm below the ionic strength for elution. Before performing preparative separations, an analytical separation should be made in order to investigate the composition of the sample. The gradient required for the preparative separation can then be evaluated from the analytical chromatogram. For a preparative separation it is important not to exceed the maximum working capacity. The higher the loading of the column, the higher flow rates and longer gradient times are to be chosen, since every single peak needs a certain volume for elution if base line separation is required. For the preparative base line separation of 100 A 60 oligo(ra) n the flow rate is increased to.5 ml/min using a gradient time of 400 min. A min For ordering information of NUCLEGEN DEAE columns please see page

37 Ion exchange columns for biochemical applications NUCLEGEN DEAE for the separation of trna, 5S RNA, viroids and messenger RNA With the development of NUCLEGEN DEAE with its pore size of 500 Å it is possible to isolate nucleic acids in the intermediate molecular weight range (5,000 1,000,000 Daltons). NUCLEGEN DEAE columns can be used for single-stranded as well as for double-stranded nucleic acids, for DNA as well as for RNA. Application with trna, 5S RNA, messenger RNA, viroids ), viral DNA and DNA restriction fragments 1) has been reported. As a consequence of their outstanding capacity and very high recovery (> 95%) the columns are particularly suitable for large-scale preparations. As some single-stranded nucleic acids can form complexes with one another, if bivalent metal ions are not excluded using EDTA (ethylenediamine tetraacetic acid), we supply columns with a PTFE inner surface coating to prevent corrosion by the EDTA. The isolated nucleic acids are pure with respect to spectroscopic, hydrodynamic and thermodynamic properties and fully active in enzymatic tests. Preparative separation of a crude RNA extract of viroid (PSTV) infected tomato plants D. Riesner BioEngineering 1 (1988) 4 48 Column: VA 15/6 NUCLEGEN DEAE 15 x 6 mm ID, Cat. No Buffer A: 50 mm KCl, 0 mm phosphate buffer ph 6.6, 5 M urea Buffer B: 1 M KCl, 0 mm phosphate buffer ph 6.6, 5 M urea Gradient: 0 50% B in 10 min % B in 50 min Flow rate: ml/min, 40 bar ambient temperature Detection: 60 nm A 60 5S RNA trna 7S RNA PSTV min NUCLEGEN DEAE for the separation of plasmids, DNA restriction fragments, ribosomal RNA, messenger RNA and viral RNA With the introduction of NUCLEGEN DEAE the scope of HPLC was extended to very high molecular weight nucleic acids (e.g megadaltons). This phase is suited for the separation of restriction fragments and oligonucleotides as well as for the separation of different ribonucleic acids. However, the load capacity is significantly lower than for the narrow-pore materials. A remarkable success of NUCLEGEN DEAE is the isolation of supercoiled plasmid DNA from a crude cell lysate in a single HPLC step 1). In a re-chromatography supercoiled forms can even be separated from the relaxed and linear forms (see chromatograms on next page). Because NUCLEGEN DEAE provides very high resolution for large as well as for medium-size nucleic acids, cdna inserts can be easily separated from the remaining vector. The plasmids are eluted from the column with more than 95% recovery and are fully active with respect to digestion with restriction enzymes, labelling with kinases, PCR, LCR reactions, transcription with polymerases and enzymatic ligation. The following buffer systems show very good separation efficiency for plasmids, viral RNA and restriction fragments. Buffer A (low salt) Buffer B (high salt) 5 M urea 1.5 M KCl or LiCl, (NH 4 ) S M K phosphate* 5 M urea ph M K phosphate*, ph % formamide, 1.5 M KCl or LiCl, (NH 4 ) S M K phosphate* 50% formamide ph M K phosphate *, ph * for stock buffer K phosphate see page 95 For ordering information of NUCLEGEN DEAE columns please see page

38 Ion exchange columns for biochemical applications Separation of plasmid pbr Sample: 5 µg plasmid pbr containing cleared lysate from E. coli Column: VA 15/6 NUCLEGEN DEAE 15 x 6 mm ID, Cat. No Eluent A: 0 mm K phosphate buffer ph 6.9; Eluent B: 5 M urea eluent A A M KCl Plasmid Gradient: 0% 100% B in 50 min arrow = ionic strength of 850 mm 0.1 RNA Flow rate: 1.0 ml/min, 70 bar, ambient temperature Detection: UV, 60 nm Separation of plasmid DNA min [M. Colpan, D. Riesner, private communication] Sample: plasmid pbr, supercoiled, relaxed and linear Column: VA 15/6 NUCLEGEN DEAE 15 x 6 mm ID, Cat. No Eluent A: 0 mm phosphate buffer ph 6.8; 6 M urea Eluent B: eluent A + M KCl Gradient: 4% 100% B in 0 min Flow rate: 1.5 ml/min, 45 bar, ambient temperature A 60 0,04 0 supercoiled relaxed linear min References 1) HPLC of high-molecular weight nucleic acids on the macroporous ion exchanger NUCLEGEN. M. Colpan, D. Riesner, J. Chromatogr. 96 (1984) 9 5 ) Large-scale purification of viroid RNA using Cs S 4 gradient centrifugation and HPLC M. Colpan, J. Schumacher, W. Brüggemann and D. Riesner, Anal. Biochem. 11 (198) ) HPLC of DNA restriction fragments. R. Hecker, M. Colpan, D. Riesner, J. Chromatogr. 6 (1985) ) HPLC zur Reinigung hochmolekularer RNAs und DNAs R. Dornburg, J. Kruppa, P. Földi, GIT 9 (1985) ) Application of HPLC technologies in rapid DNA sequencing of kilobase pairs of DNA R. Dornburg, LC. GC 6 (1988) ) Fractionation of DNA restriction fragments with ion exchangers for HPLC W. Müller, Eur. J. Biochem. 155 (1986) 0 1 7) Isolation of high-molecular nucleic acids for copy number analysis using HPLC. S. J. Coppella, C. M. Acheson, P. Dhurjati, J. Chromatogr. 40 (1987) ) Chromatographic separation of DNA restriction fragments (Review). R. Hecker, D. Riesner, J. Chromatogr. 418 (1987) Separation of DNA restriction fragments Sample: 5 µg DNA restriction fragments of plasmid psp 64, digested with Hinf I Column: VA 15/6 NUCLEGEN DEAE, 15 x 6 mm ID, Cat. No Gradient: linear, 50 mm 150 mm KCl in 00 min, in 0 mm K phosphate buffer ph 6.5, k 5,5 M urea Flow rate: 1 ml/min i Temperature: C Detection: UV, 60 nm Fragment lengths: a) 19 bp b) 5 bp c) 8 bp d) 41 bp e) 45 bp f) 78 bp g) 168 bp h) 96 bp i) 4 x 44 bp j) 71 bp k) 881 bp a b cde f min g h j 98

39 Ion exchange columns for biochemical applications NUCLESIL PEI anion exchanger for the separation of proteins and peptides The purification of biologically active peptides and proteins becomes more and more important in research and industry. Ion exchange chromatography is a superior technique in this regard, because it allows purification of proteins in aqueous buffers using salt or ph gradients. The low isoelectric point of most proteins is the reason why anion exchange chromatography is preferably applied. The 400 nm pores of the rigid silica matrix of NUCLESIL PEI enable an unrestricted penetration of polypeptides under very different elution conditions resulting in short analysis times. The mechanical stability of the spherical microparticles of this packing allow high flow rates and a long-term column life. The polymeric, covalently bonded polyethylene imine network guarantees a good chemical stability of the columns towards basic and acidic eluents. The coating is flexible enough to fit different protein shapes for the formation of specific electrostatic interactions. Characteristic parameters of the column packing: Type: weakly to strongly basic anion exchanger Mean particle size: 7 µm Mean pore size: 400 nm ph range: 8.5 Restrictions for the use of none buffers: Chemical stability with acids good or bases: Restrictions for the use of none organic solvents: Maximum salt concentration: 8 M Ion exchange capacity: 0.15 mmol/g Protein binding capacity: 61 mg BSA/g Maximum working pressure: 50 bar Length 50 mm 15 mm 50 mm Guard columns NUCLESIL PEI polyethyleneimine network, covalently polymer-coated onto NUCLESIL silica, weakly basic anion exchanger, particle size 7 ± 1.5 µm, pore size 4000 Å; eluent in column methanol EC analytical columns 1) 4 mm ID Standard-Prep preparative columns 10 mm ID Standard columns are manufactured from stainless steel. Metal-free columns (PEEK) can be custom-packed with 4.6 or 7.5 mm ID and lengths of 50, 150 and 50 mm. ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). NUCLESIL PEI shows a high selectivity for numerous proteins, e.g β-lactoglobulins A and B, two proteins differing in just two amino acids, can be separated in only 10 minutes. Most of the proteins which can be purified on NUCLESIL PEI are obtained with high yields, preserving their biological activity. Recovery of enzyme activity conditions Columns: EC 50/4 NUCLESIL PEI, 50 x 4 mm ID, Cat. No Buffers: A) 0 mm Tris/HCL ph 8.5; B) A M NaCl Gradient: 0 100% B in 5 min, 1 ml/min, 0 bar The eluting peaks were detected and fractionated by their UV absorbance at 80 nm. The specific enzyme activity was estimated before and after HPLC by comparison of the UV spectra of the proteins with their volume activity. Enzyme Recovery of specific activity after HPLC [%] Catalase (bovine liver) 9 L-Lactic dehydrogenase LDH-1 10 isoenzyme (porcine heart) Callicrein (porcine pancreas) 98 Glucose oxidase 104 (Aspergillus niger) Peroxidase (horseradish)

40 Ion exchange columns for biochemical applications Recovery of proteins Column: EC 50/4 NUCLESIL PEI, 50 x 4 mm ID, Cat. No Eluent: 10 mm NaH P 4, 1.5 M NaCl, ph 7.0 Flow rate: 1 ml/min Sample: 50 µg of each protein Protein Yield [%] Myoglobin 100 Transferrin 95 valbumin 98 Bovine serum albumin 100 Glucose oxidase 100 α-amylase 100 Soybean trypsin inhibitor 100 β-lactoglobulin 97 Ferritin 85 Separation of a monoclonal antibody from undialysed ascites fluid 46. mg/ml of total protein, sample volume 5 µl Column: EC 15/4 NUCLESIL PEI, 15 x 4 mm ID, Cat. No Eluent A: 10 mm TRIS/HCl ph 8. Eluent B: eluent A M NaCl Gradient: 0 1% B in 5 min, then 1 70% B in 0.5 min Flow rate: 1 ml/min Pressure: 50 bar Detection: UV, 80 nm IgG b The flexible anion exchange groups of NUCLESIL PEI also show good binding and desorption kinetics for nucleotides, as is shown in the figure below. Anion exchange chromatography of nucleotides a) Separation of 9 nucleotides Column: EC 15/4 NUCLESIL PEI, 15 x 4 mm ID, Cat. No Eluent A:.5 mm TRIS/phosphate ph 7. Eluent B:.5 mm TRIS/phosphate ph M KCl Gradient: 5 95% B in 5 min Flow rate: 1. ml/min Pressure: 10 bar Detection: UV, 60 nm b) Separation of adenine nucleotides in 90 seconds Column: EC 50/4 NUCLESIL PEI, 50 x 4 mm ID, Cat. No Eluent A: 0 mm TRIS/acetate ph 8.0 Eluent B: eluent A M NaCl Gradient: 0 80% B in 0 s Flow rate: ml/min Pressure: 85 bar Detection: UV, 60 nm Peaks: 1. CMP. AMP 7. GMP 4. CDP a) b) 5 5. ADP 6. GDP 9 7. CTP ATP 9. GTP min s min This silica-based exchanger can be cleaned from sticking impurities by a 10-min treatment with 0.1 M NaH or 1 M HN. 100

41 Ion exchange columns for biochemical applications peration of NUCLESIL PEI columns Separation principle Separation is obtained by reversible adsorption of negatively charged substances to positively charged groups on the exchanger material and their subsequent displacement by either increasing ionic strength or ph changes in the mobile phase. Mobile phase In order to provide efficient adsorption of a protein to the anion exchanger it is usually preferable to select a ph value about one unit above the isoelectric point of the protein. The nature of the counterion is an important aspect to achieve high resolution. By using different buffers and/or elution salts (e.g. sodium acetate, ammonium acetate, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate) one can easily change the ionic conditions of the separation process. This procedure can influence the sequence of elution and the peak shape. Divalent cations (e.g. Mg ++, Ca ++ ) usually show a stronger eluting power than monovalent ones. When using a salt gradient most of the sample compounds are eluted from the column at moderate salt concentrations ( 1 mol/l). If the compound of interest cannot be eluted a change of ph and/or buffer and salt ions should be considered. For difficult cases non-ionic detergents or organic solvents (e.g. 5% acetonitrile) will increase the solubility and desorption of the polypeptide. rganic modifiers should be premixed with the aqueous buffer to avoid precipitation of buffer. The eluents should be filtered through 0.45 µm filters in order to prevent the accumulation of particulate matter. Solvents should be degassed to ensure a continuous flow through the system. Column installation and operation The column is supplied with methanol. Use distilled water as initial solvent to prepare the column for use with aqueous salt buffers. Equilibration with the starting buffer is finished, when the signal of the detector has reached a stable value. Avoid sudden pressure surges since they may destroy the column packing. If possible, dissolve the sample in the starting buffer and filter through a 0.45 µm filter. Column cleaning and storage NUCLESIL PEI columns can easily be cleaned from sticking impurities by several hours of treatment with M sodium acetate ph 8.5, short incubation (10 min, 5 column volumes) with 0.1 M NaH, 1 M HN, 1% CH CH or by using organic solvents like acetonitrile, methanol or dimethylsulphoxide. Application of non-ionic detergents like Triton X 100 or chaotropic reagents like guanidinium hydrochloride or urea is also possible. vernight columns should be stored in 0.05% sodium azide / water or methanol/water (80:0, v/v). For a longer storage all salts have to be washed out from the columns, which are then equilibrated with methanol. Examples for the purification of different peptides and proteins can be found in our catalogue LC Applications or on the internet. For ordering information of NUCLESIL PEI columns please see page Separation of protein standards Column: EC 15/4 NUCLESIL PEI, 15 x 4 mm ID, Cat. No Eluent A: mm TRIS/acetate ph 8.0 Eluent B: 0 mm TRIS/acetate ph M KCl Gradient: linear 0 40% B in 0 min Flow rate: 1 ml/min Pressure: 76 bar Detection: UV, 80 nm Peaks: (Volume 0 µl) 1. Catalase. Myoglobin. α-amylase 6 4. Transferrin 5. α-lactalbumin 7 6. Glucose oxidase 7. Soybean trypsin inhibitor min Tryptic digest of bovine serum albumin Sample volume 50 µl Column: EC 15/4 NUCLESIL PEI, 15 x 4 mm ID, Cat. No Eluent A: 0 mm TRIS/acetate ph 8 Eluent B: A) + 1 M KCl Gradient: 0 0% B in 0 min, then 0 80% B in 10 min Flow rate: 0.5 ml/min Pressure: 8 bar Detection: UV, 80 nm min

42 Ion exchange columns for biochemical applications NUCLEGEL SAX strongly basic anion exchanger for biological macromolecules Separation of hen s egg white Sample: frozen egg white was thawed, filtered and diluted 1:8 with eluent A Injection: 50 µl Column: VA 50/4.6 NUCLEGEL SAX , 50 x 4.6 mm ID, Cat. No Eluent A: 0.01 M Tris HCl, ph 7.5 Eluent B: A + 0,5 M NaAc, ph 7.5 Gradient: linear, 0 100% B in 0 min Flow rate: 1 ml/min Detection: UV, 80 nm Peaks: 1. Conalbumin. valbumin. not identified min This column family features a fully quaternised polyethyleneimine structure coupled to macroporous hydrophilic polymer beads. Pore sizes of 100 and 400 nm, respectively, allow the purification of peptides, large proteins and oligonucleotides. This strongly basic anion exchanger possesses a high capacity for proteins even at ph 10; for this reason it can be applied for difficult protein separations in the ph range around and above 10. Due to the polymer matrix, ph stability of the exchanger is excellent, allowing removal of pyrogens by washing with 0.1 M NaH over several hours. Characteristic parameters of the column packing Type: strongly basic anion exchanger ph range: 1 1 restrictions for the use of buffers: none (polar organic solvents can be used as modifiers) maximum salt concentration: 8 M ion exchange capacity: > 0. mmol/g protein binding capacity: 40 or 0 mg BSA/g, respectively maximum operating pressure: 00 bar Length 50 mm 150 mm Guard columns NUCLEGEL SAX strong anion exchanger -N(CH ), polymer-based, gel matrix quaternised PEI; particle size 8 µm, pore size 1000 Å; eluent in column 0.1 M Na S % NaN Valco type analytical columns 1) 4.6 mm ID mm ID NUCLEGEL SAX strong anion exchanger as above, particle size 10 µm, pore size 1000 Å Standard-Prep preparative columns 5 mm ID NUCLEGEL SAX strong anion exchanger as above, particle size 8 µm, pore size 4000 Å Valco type analytical columns 1) 4.6 mm ID mm ID NUCLEGEL SAX strong anion exchanger as above, particle size 10 µm, pore size 4000 Å Standard-Prep preparative columns 5 mm ID Columns are supplied in packs of 1. ) Valco type guard column cartridges are 5 x mm, require guard column holder B (Cat. No ) and are supplied in packs of. 10

43 Ion exchange columns for biochemical applications NUCLEGEL SCX strongly acidic cation exchanger for biological macromolecules Cation exchangers are an important supplement for the ion chromatographic purification of biological macromolecules. Especially for proteins, peptides and carbohydrates with high isoelectric points this technique is a valuable tool. The macroporous structure of NUCLEGEL SCX columns guarantees unhindered contact of large molecules with the exchange functionalities. Complex samples (e.g. cell extracts) or solubilised membrane protein mixtures often pose problems because they cannot be recovered quantitatively from the column. In this case, the polymer matrix of the NUCLEGEL SCX columns allows use of strongly basic or acidic eluents for an effective column regeneration. Characteristic parameters of the column packing Type strongly acidic cation exchanger ph range 1 1 restrictions for the use of buffers none (polar organic solvents can be used as modifiers) maximum salt concentration 8 M maximum working pressure 00 bar Separation of protein standards Column: VA 50/4.6 NUCLEGEL SCX , 50 x 4.6 mm ID, Cat. No Eluent A: 0.0 M KH P 4, ph 6.0 Eluent B: A M NaCl, ph 6.0 Gradient: linear, 0 100% B in 0 min Flow rate: 1 ml/min Detection: UV, 80 nm 1 Peaks: 1. Myoglobin. α-chymotrypsinogen A. Cytochrome C 4. Lysozyme min Length 50 mm 150 mm Guard columns NUCLEGEL SCX strong cation exchanger - S, hydrophilic gel matrix; particle size 8 µm, pore size 1000 Å; eluent in column 0.1 M Na S % NaN Valco type analytical columns 1) 4.6 mm ID mm ID NUCLEGEL SCX strong cation exchanger as above, particle size 10 µm, pore size 1000 Å Standard-Prep preparative columns 5 mm ID NUCLEGEL SCX strong cation exchanger as above, particle size 8 µm, pore size 4000 Å Valco type analytical columns 1) 4.6 mm ID mm ID NUCLEGEL SCX strong cation exchanger as above, particle size 10 µm, pore size 4000 Å Standard-Prep preparative columns 5 mm ID Columns are supplied in packs of 1. 1) Valco type guard column cartridges are 5 x mm, require guard column holder B (Cat. No ) and are supplied in packs of. 10

44 Reversed phase columns for biochemical applications Reversed phase chromatography (RPC) is increasingly used as an efficient method in peptide and protein analysis. It gains increasing importance in analytical biochemistry for the purification of clinically relevant, genetically engineered polypeptides. With RPC often small changes in the hydrophobic surface regions of peptides and proteins can be detected, such as e.g. exchange of an amino acid or the presence of deamidation or oxidation products. During workup of industrial scale preparations RPC is increasingly applied for the final purification. Examples include biosynthetic proteins such as human growth hormone, tissue plasminogen activator, human insulin and human malaria vaccine. Characteristic parameters of the RP phases Packing ph working range max. working pressure [bar] For seven proteins tested the mass recovery for all phases is %. Separation principle Substances with hydrophobic surface regions are reversibly bonded to the hydrophobic alkyl chains of the RP stationary phase. By increasing the concentration of the organic component in the eluent, the hydrate shell of the molecule is decreased resulting in desorption and chromatographic separation of substances according to their hydrophobicity. For biological macromolecules partition effects have no significance. We offer three different types of RP columns for biochemical applications: NUCLESIL MPN alkyl chains monomerically bonded to silica NUCLESIL PPN alkyl chains polymerically bonded to silica NUCLEGEL RP polymer-based phases Due to their outstanding performance NUCLESIL reversed phase materials are very well suited for these applications. We offer a complete RPC column family for analytical as well as for preparative separations. The silica- and polymer-based stationary phases differ significantly with respect to their surface structure and their chromatographic properties. dynamic protein binding capacity mg protein per g packing BSA cytochrome C Silica-based phases NUCLESIL C 18 MPN NUCLESIL 00-5 C 18 MPN NUCLESIL 00-5 C 4 MPN NUCLESIL C 18 PPN NUCLESIL C 18 PPN NUCLESIL C PPN Polymer-based phases NUCLEGEL RP C NUCLEGEL RP NUCLEGEL RP NUCLEGEL RP As can be seen from the left figure below, the maximum separation efficiency can be achieved when the injected protein mass does not exceed 1 % of the maximum protein loading capacity. In practice often sufficient chromatographic resolution is obtained for higher injection masses. The sample volume does not influence the separation result over a wide range. For this reason RP chromatography often results in a concentration of the biomolecule (see right figure). Peak volume µl Mass sensitivity µg Protein mass (Cytochrome C) EC 15/4 NUCLESIL C 18 PPN, Cat. No , eluent: A) 0.1% TFA in H, B) 0.08% TFA in CH CN Chromatographic resolution as a function of sample volume EC 15/4 NUCLESIL C PPN, Cat. No , eluent A) 0.1% TFA in H, B) 0.08% TFA in CH CN, gradient 0% 60% B in 10 min, proteins cytochrome C, bovine serum albumin chromatographic resolution R s µl sample volume 104

45 Reversed phase columns for biochemical applications Columns based on NUCLESIL for the separation of proteins/peptides and oligonucleotides NUCLESIL MPN This type of RP phases is based on NUCLESIL silica monomerically bonded with alkyl chains. This flexible brush-type structure guarantees high selectivities. The predominantly hydrophobic forces are supplemented by a small portion of hydrophilic interactions. This is a prerequisite for the successful separation of e.g. peptides with equal molecular weight but different net charges. The µm silica with C 18 modification shows an outstanding selectivity for peptides, while the wide pore C 4 material is especially suited for the purification of larger, hydrophobic peptides and very different proteins. If possible the columns should be used in the ph range between and 8. Column: Eluent A: Eluent B: Gradient: Flow rate: Detection: Peaks: 1. Hem. β-globin. α-globin 4. A γ T -globin 5. G γ-globin 6. A γ I -globin Separation of hemoglobin chains EC 50/4 NUCLESIL 00-5 C 4 MPN 50 x 4 mm ID, Cat. No % acetonitrile, 80% water, 0.1% TFA 60% acetonitrile, 40% water, 0.1% TFA from 40 to 60% B in 60 min 1 ml/min UV, 0 nm 1 5 Absorbance, 0 nm min Length 50 mm 15 mm 50 mm Guard columns EC analytical columns 1) NUCLESIL C 18 MPN ctadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column methanol mm ID mm ID NUCLESIL 10- C 18 MPN ctadecyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size ~.5 µm, pore size 10 Å; eluent in column methanol mm ID mm ID NUCLESIL 00-5 C 4 MPN Butyl phase, alkyl chains monomerically bonded to silica, brush type structure, particle size 5 ± 1.5 µm, pore size 00 Å; eluent in column methanol mm ID mm ID The above columns are manufactured from stainless steel. Metal-free columns (PEEK) with 4.6 or 7.5 mm ID and lengths of 50, 150 and 50 mm can be custom-packed on request. ChromCart guard column cartridges (8 mm) in packs of, all other cartridges in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). 105

46 Reversed phase columns for biochemical applications Columns based on NUCLESIL for the separation of proteins/peptides and oligonucleotides NUCLESIL PPN These columns are packed with silica with a polymeric alkyl modification on NUCLESIL silica. The polymeric layer ensures that peptides and proteins are exclusively retained by hydrophobic forces. Polypeptides which differ in shape and size of their hydrophobic surface regions are separated. Thus these columns feature different selectivities compared to the MPN material, a fact which may improve the solution of certain separation problems. With NUCLESIL C 18 PPN good peak shapes are also found for basic peptides. The wide pore NUCLESIL PPN phases are available in two different alkyl chain lengths (octadecyl and propyl), which differ in hydrophobicity and rigidity of the alkyl network. While the C 18 material is especially suited for large peptides and medium-size hydrophilic proteins, the C phase is recommended for larger and hydrophobic proteins. Memory effects are reduced. The PPN columns can be used with eluent containing organic bases up to ph 9.5. Length 50 mm 15 mm 50 mm Guard columns NUCLESIL C 18 PPN ctadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 100 Å; eluent in column methanol EC analytical columns 1) mm ID mm ID Standard-Prep preparative columns 10 mm ID NUCLESIL C PPN Propyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 500 Å; eluent in column methanol EC analytical columns 1) mm ID mm ID Standard-Prep preparative columns 10 mm ID NUCLESIL C 18 PPN ctadecyl phase, alkyl chains polymerically bonded to silica, particle size 5 ± 1.5 µm, pore size 500 Å; eluent in column methanol EC analytical columns 1) 4 mm ID Standard-Prep preparative columns 10 mm ID The above columns are manufactured from stainless steel. Metal-free columns (PEEK) with 4.6 or 7.5 mm ID and lengths of 50, 150 and 50 mm can be custom-packed on request. Corresponding VarioPrep columns can also be supplied on request. ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of 1. 1) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7159). Amount of NUCLESIL packing per column dimension for MPN and PPN phases: 50 x 4 mm 0.4 g 15 x mm 0.15 g 15 x 4 mm 0.6 g 15 x 10 mm.6 g 50 x mm 0. g 50 x 4 mm 1. g 50 x 10 mm 7. g 106

47 Reversed phase columns for biochemical applications The polymeric coating of NUCLESIL C 18 PPN shows outstanding stability at alkaline ph and the absence of nonspecific interactions with alkaline or acidic compounds. Therefore, this column is suited for the separation of peptides and proteins up to about 40 kd. The column may be purified by treatment with Si -saturated NaH. 10 to 0 column volumes of the alkaline eluent should be enough to wash away sticking impurities from the column. This procedure will have a sterilising effect, too. Retention times, peak shapes and mass recoveries are not affected by passing more than 00 column volumes of the alkaline eluent through the column as is shown on the right. Separation of pancreatic secretion of piglets Column: EC 15/4 NUCLESIL C 18 PPN 15 x 4 mm ID, Cat. No Eluents: A) 0.1% TFA in H, B) 0.08% TFA in CH CN 1 Gradient: linear 0 50% B in 14 min, then 50 65% B in 6 min Flow rate: 1 ml/min Detection: UV, 15 nm Peaks: 1. Trypsin + trypsinogen. Proelastase. Lipase + α-chymotrypsin 4. Chymotrypsinogen 5. α-amylase 6., 7. Procarboxypeptidase min Separation of commercial bacitracin Column EC 15/4 NUCLESIL C 18 PPN 15 x 4 mm ID, Cat. No Eluent A: 0.1% TFA in H Eluent B: 0.08% TFA in CH CN Gradient: linear 0 40% B in 15 min Flow rate: 1 ml/min Detection: UV, 15 nm Separation of protein standards left chromatogram: before treatment, right chromatogram: after treatment with 00 column volumes of the alkaline eluent (= 1 volume 50 mm NaH + 1 volume n-propanol, saturated with Si by stirring the mixture with 1 g/l unmodified silica gel for 1 day) at a flow rate of 0.4 ml/min Column: EC 15/4 NUCLESIL C 18 PPN, 15 x 4 mm ID, Cat. No Eluent A: A) 0.1% TFA in H, B) 0.08% TFA in CH CN Gradient 0 60% B in 10 min Flow rate: 1.0 ml/min Detection: UV, 80 nm Peaks: 1. Ribonuclease. Cytochrome c. Lysozyme 4. β-lactoglobulin 5. valbumin 0 min 0 min Separation of a 0mer oligonucleotide (Sample courtesy of Dr. Essrich, Inst. Prof. Seelig, Karlsruhe, Germany) Column EC 50/4 NUCLESIL C 18 PPN, 50 x 4 mm ID, Cat. No Eluent A: 0.1 M triethylammonium acetate ph 7.0 acetonitrile (95:5, v/v) Eluent B: 0.1 M triethylammonium acetate ph 7.0 acetonitrile (0:70, v/v) Gradient: linear 15 40% B in 0 min Flow rate: 1 ml/min Detection: UV, 90 nm min min 107

48 Reversed phase columns for biochemical applications Polymer-based RP columns NUCLEGEL RP C 18 columns These polymer-based columns have been developed for reversed phase chromatography in the ph range from 1 to 14. They contain a C 18 modified polystyrene-divinylbenzene polymer with outstanding stability, inertness and improved peak symmetry even for basic substances. nly simple eluent systems are required for the separation. Due to improved sensitivity post-column derivatisations are seldom necessary. NUCLEGEL RP columns This type of reversed phase column is based on a polystyrene resin cross-linked with DVB. Due to the excellent stability of the particles compared to other resins operation under reversed phase conditions is possible. The ph range applicable with these columns reaches from ph 1 to ph 1. The small pore columns for reversed phase separation of small molecules are especially suited for pharmaceutical compounds with basic properties, such as organic heterocycles. They can also be used for the separation of nucleosides and nucleotides up to 5000 dalton and allow gradient as well as isocratic elution. The wide pore columns are especially recommended for large biomolecules. For proteins they show good peak shapes and selectivities. Compared to a silica matrix the higher background hydrophobicity of the NUCLEGEL RP phases can be disadvantageous for the mass recovery of some large hydrophobic proteins. The peak capacity for the separation of complex peptide mixtures is lower than for NUCLESIL MPN or PPN. A working pressure of 180 bar should not be exceeded. Separation of sympathomimetic amines Column: VA 150/4.6 NUCLEGEL RP C 18, 150 x 4,6 mm, Cat. No Eluent A: 0.1 M ammonia acetonitrile (68 : ) Eluent B: 0.1 M ammonia acetonitrile (54 : 46) Gradient: linear 0 100% B in 5 min Flow rate: 1 ml/min Temperature: ambient 1 Detection: UV, 0 nm Peaks (0 µl injected): 1. Phenylpropanolamine. Pseudoephedrine. Amphetamine 4. Methamphetamine 4 Separation of cephalosporin antibiotics Column: VA 150/4.6 NUCLEGEL RP C 18, 150 x 4,6 mm, Cat. No Eluent: 0.1% aqueous tetrabutylammonium bromide solution acetonitrile (55 : 45) Flow rate: 0.5 ml/min Detection: UV, 54 nm 1 Peaks: 1. Cefalexin. Cefalotin. Cefazolin 4. Cefaloridine min min 108

49 Reversed phase columns for biochemical applications Polymer-based RP columns ordering information NUCLEGEL RP C 18 Length 50 mm 15 mm 150 mm 50 mm 00 mm Guard columns C 18 modified PS/DVB polymer for RP separations in the ph range 1 14; pore size 80 Å; eluent in column CH CN/H Valco- type analytical columns 1) 4.6 mm ID particle size 10 µm NUCLEGEL RP 100 Polystyrene resin cross-linked with divinylbenzene (PS/DVB); pore size 100 Å; eluent in column CH CN/H Valco type analytical columns 1) 4.6 mm ID particle size 5 µm mm ID particle size 8 µm mm ID particle size 8 µm Standard-Prep preparative columns 5 mm ID particle size 10 µm NUCLEGEL RP 00 Polystyrene resin cross-linked with divinylbenzene (PS/DVB); pore size 00 Å; eluent in column CH CN/H Valco type analytical columns 1) 4.6 mm ID particle size 5 µm mm ID particle size 8 µm mm ID particle size 8 µm Standard-Prep preparative columns 5 mm ID particle size 10 µm NUCLEGEL RP 1000 Polystyrene resin cross-linked with divinylbenzene (PS/DVB); pore size 1000 Å; eluent in column CH CN/H Valco type analytical columns 1) 4.6 mm ID particle size 8 µm mm ID particle size 8 µm Standard-Prep preparative columns 5 mm ID particle size 10 µm NUCLEGEL RP 4000 Polystyrene resin cross-linked with divinylbenzene (PS/DVB); pore size 4000 Å; eluent in column CH CN/H Valco type analytical columns 1) 4.6 mm ID particle size 8 µm mm ID particle size 8 µm Standard-Prep preparative columns 5 mm ID particle size 10 µm Columns are supplied in packs of 1. 1) Valco type guard column cartridges are 5 x mm, require guard column holder B (Cat. No ) and are supplied in packs of. 109

50 Reversed phase columns for biochemical applications Quantitative determination of vancomycin Concentration and decomposition of the glycopeptide antibiotic vancomycin can be quantitatively monitored with NUCLEGEL RP columns. The figure shows the chromatogram of a vancomycin sample after 15 years at 56 C. Column: VA 150/4.6 NUCLEGEL RP 100-8, 150 x 4,6 mm ID, Cat. No Eluent A: 8% (v/v) acetonitrile 0.0 M borate buffer, ph 8.0 Eluent B: 16% (v/v) acetonitrile 0. M borate buffer, ph AU Gradient: linear, 0 100% B in 17.5 min Flow rate: 0,5 ml/min Detection: UV, 5 nm Separation of proteins Column: VA 50/4.6 NUCLEGEL RP 00-8, 50 x 4,6 mm ID, Cat. No Eluent A: 0.1% TFA in acetonitrile water (95:5) Eluent B: 0.1% TFA in water Gradient: linear, 0 60% A in min Flow rate: 1,5 ml/min 4 Detection: UV, 0 nm 6 Peaks: 1. Ribonuclease A. Insulin 5. Cytochrome C 4. Lysozyme 5. Bovine serum albumin 6. Myoglobin 7. Hen s egg white 1 7 min peration of reversed phase columns Mobile phase: Besides selection of the column, choice of the eluent is very important for the success of a chromatographic separation. Adsorption of the biological macromolecules is usually achieved from aqueous buffer solutions. Additives often applied are % trifluoroacetic acid or phosphoric acid in acidic medium and pyridine/formate or ammonium acetate in the neutral ph range. These ion pairing reagents can either decrease or increase the polarity of the peptide. As organic modifiers, mainly acetonitrile, 1-propanol or -propanol are used. In this series, the elution strength of the solvents increases. ften a good selectivity can be obtained with ternary systems, e.g. water / acetonitrile / 1-propanol. Biological macromolecules are almost exclusively obtained by gradient elution. Column installation and operation: Columns should first be washed with 4 column volumes of water. Then the column can be equilibrated with the corresponding buffer system. Abrupt flow and pressure surges should be avoided, because they can destroy the column packing. If possible, samples are dissolved in the starting buffer and filtered through a 0.45 µm filter. If you have to separate an unknown sample mixture, and if there is no similar separation known from literature, you can choose the chromatographic test conditions of the column as a first attempt. Column cleaning and storage: All columns described in this chapter can be cleaned with several 10 0 min gradients from TFA/water to 80% acetonitrile or 60% -propanol. Addition of 0.1 M EDTA can be of advantage in some cases. If possible, the column should be cleaned from uneluted sample components after each day of use. With NUCLE- GEL columns, organic impurities can also be removed by flushing with 0.1 M NaH. The columns should be stored in an acid-free eluent with a high percentage of organic solvent, e.g. 70% methanol. For separations of different peptides, proteins or nucleic acids with these columns please see our catalogue LC Applications or visit our website: 17 min

51 Reversed phase columns for biochemical applications References: 1) Automated evaluation of tryptic digest from recombinant human growth hormone using UV spectra and numeric peak information H.-J. P. Sievert et al. J. Chrom. 499 (1990) 1 4 ) Separation and quantitative determination of high molecular-weight subunits of glutenin from different wheat varieties and genetic variants of the variety sicco W. Seilmeier, H.-D. Belitz, H. Wieser, Z. Lebensm. Unters. Forsch. 19 (1991)14 19 ) Isocratic separation of phenylthiohydantoinamino acids by RP-HPLC K. Hayakawa, J. izumi, J. Chrom. 487 (1989) ) Rapid method based on RP-HPLC for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides G. Giegerich, M. Pette, K. Fujita, H. Wekerle, J. T. Epplen, A. Hinkkanen, J. Chrom. 58 (1990) ) Non-ideal behaviour of silica-based stationary phases in TFA-acetonitrile-based RP-HPLC separations of insulins and proinsulins S. Linde, B. S. Welinder, J. Chrom. 56 (1991) ) Effects of eluent composition, ion pair reagent and temperature on the separation of histones by HPLC H. Lindner, W. Heiliger, Chromatographia 0 (1990) Rapid reversed phase separation of proteins Column: VA 50/4.6 NUCLEGEL RP , 50 x 4,6 mm, Cat. No Eluent A: 0.1% TFA in CH CN H (5 : 95, v/v) Eluent B: 0.1% TFA in CH CN H (95 : 5, v/v) Gradient: linear, 18-60% B in 60 seconds Flow rate: 4 ml/min Detection: UV, 80 nm 4 Peaks (total protein 0.4 mg) 1. Ribonuclease A. Cytochrome C. Lysozyme 4. Bovine serum albumin 5. Myoglobin 5 6. valbumin s Rapid reversed phase separation of peptides Column: VA 50/4.6 NUCLEGEL RP , 50 x 4,6 mm, Cat. No Eluent A: 0.1 % TFA in acetonitrile water (1 : 99) Eluent B: 0.1 % TFA in acetonitrile water (99 : 1) Gradient: linear, 10 60% B in min Flow rate: 4 ml/min Detection: UV, 0 nm Peaks: Neurotensin fragment 1-8. Neurotensin fragment 8-1. Neurotensin 4. Myoglobin s 111

52 Gel filtration columns for biochemical separations Gel filtration chromatography (GFC) as a special form of size exclusion chromatography requires pressure-stable high performance columns for the separation of biopolymers according to size and shape. NUCLESIL GFC silica-based columns NUCLESIL GFC is a silica-based column with a hydrophilic polyalcohol modification. MACHEREY-NAGEL offers HPLC columns for GFC based on silica or polymer resins. Characteristic parameters of the column packing Working range for globular proteins ph working range 8 minimum salt concentration 100 mm polar organic solvents (modifiers) 0 100% maximum pressure 100 bar typical flow rate ml/min typical temperature 10 0 C Length 00 mm Guard columns NUCLESIL 15-5 GFC hydrophilic polyalcohol modification on silica; particle size 5 µm, pore size 15 Å; eluent in column 0.1 M NaH P 4 ph 6.7 / 0.1 M Na S 4 (1:1, v/v) % NaN Valco type analytical columns 7.7 mm ID Columns are supplied in packs of 1. ChromCart guard column cartridges CC 0/4 NUCLESIL 15-5 GFC are 0 mm long and supplied in packs of. They require the CC column holder 0 mm (Cat. No. 718). Separation of standard proteins Column: VA 00/7.7 NUCLESIL 15-5 GFC, 00 x 7.7 mm ID, Cat. No Eluent: 0.05 M NaH P 4, 0. M NaCl, ph 7.0 Flow rate: 1 ml/min Detection: UV, 0 nm Peaks: 1. Thyroglobulin. γ-globulin. valbumin 4. RNase A 5. p-aminobenzoic acid 4 5 Separation of standard proteins Column: VA 00/7.7 NUCLESIL 15-5 GFC, 00 x 7.7 mm ID, Cat. No Eluent: 0.0 M NaH P 4, 0. M NaCl, ph 7.0 Flow rate: 1 ml/min Detection: UV, 0 nm Peaks: 1. Ferritin. BSA. β-lactoglobulin 4. Cytochrome C 5. Uridine Separation of hemoglobin and myoglobin Column: VA 00/7.7 NUCLESIL 15-5 GFC, 00 x 7.7 mm ID, Cat. No Eluent: 0.1 M NaH P 4, 0. M NaCl, ph 7.0 Flow rate: 1 ml/min Detection: UV, 80 nm Peaks: 1. Hemoglobin. Myoglobin min min min 11