Products for GPC. Products for GPC. Size exclusion chromatography. Basic principles

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1 Products for GPC Products for GPC Size exclusion chromatography Basic principles injection A B C retention time mobile phase B Size exclusion chromatography (SEC) is a liquid chromatographic separation method which permits the analysis of molecules in the oligomer and polymer range. This method is also known as gel permeation chromatography (GPC). The main difference between SEC or GPC and other chromatographic methods is that a separation is not achieved through any kind of interaction but quite simply by classifying the molecules by size. Separation mechanism. The chromatographic column is packed with particles of the same size (in the case of NUCLEOGEL columns particles are spherical with diameters of 5, 8 or 10 µm). For the classification of molecules according to size it is a prerequisite that the particles are totally porous and that the pores have a defined diameter. In the figure above the pores are illustrated in a conical shape for demonstration purposes. Let us assume, that the polymers to be analysed consist of molecules size A, B and C. In the example illustrated, the largest molecules A cannot enter the pores, while molecule B can only half enter and the smallest molecules C can completely enter the pore system. Since adsorptive interactions with the stationary phase can be excluded in GPC by correct choice of the mobile phase, elution of the components is in the order A, B and C, i. e. the largest molecules are eluted first and the smallest last. In order to achieve an optimum separation of oligomers or polymers, packing materials with different pore diameters are available. These are characterised by pore size or exclusion limit. The exclusion limit corresponds to the molecular weight of the polystyrene or dextran fraction which can just enter the gel and is thereby a measure for the pore size distribution of the gel. A B C column packing C pores Illustration of the separation mechanism in GPC Based on the previous explanations the following conclusions can be drawn: All injected molecules are eluted again. The chromatogram is complete when the dead time is reached, thus it is known in advance how long the separation will take. The elution volume or elution time only depends on the size of the molecule, and thereby indirectly on its molar mass. SEC (GPC) can be used to determined molecular weights. Since the separation is complete after a volumetric flow corresponding to just the pore volume of the column, the peak capacity (i.e. the number of peaks which can be separated with a definite resolution) is limited. In order to separate two sizes of molecules, there should be a difference in their molecular mass of at least 10 %. Since the retention of the molecules in the stagnant mobile phase is not only desirable but encouraged (as opposed to other chromatographic methods), the resultant theoretical plate numbers are generally not as high as otherwise in normal HPLC. Since the elution volumes are small, minimising the dead volume in the complete chromatographic system is very important. The following figure again illustrates the separation mechanism. a b c Schematic illustration of GPC in three steps Test molecules which are too large to enter the pores can only be dispersed through the volume between individual beads of the packing material: they are excluded and must find their way through the column in the space between the spherical particles. Since there are no interactions between the sample and the stationary phase, these molecules are carried through the column by the mobile phase and eluted as fast as possible. On the other hand, if small molecules are present which can completely enter the pores, the total volume of the mobile phase is at their disposal. Since the mobile phase is stagnant in the pores, the molecules in the pores are moved only by diffusion and are retained longer than the excluded molecules. They appear in the detector as the last peak, and in fact exactly at the dead time known from other column chromatographic methods. 122

2 Products for GPC Products for GPC Size exclusion chromatography Molecules of medium size cannot enter the narrowest pores and only partially permeate the stationary phase. At their disposal is a volume between the total permeation and the dead volume. They are eluted after the excluded molecules, but before the dead time. Calibration chromatogram. If one wants to know which elution volume V E corresponds to a definite molecular size, the column is first calibrated with a test mixture composed of components with exactly known molecular weights. The size of the calibration molecules (polymer standards) must be chosen such that one component is excluded, several components partially enter the pores, one component totally permeates the stationary phase. Calibration standards for organic GPC systems are mainly defined polystyrenes, and for aqueous separation systems dextran standards with different, but defined molecular weights are used. For our programme of polymer standards please see pages 131 and following. V z exclusion volume C 4 H 9 CH 2 CH H The figure shows a chromatogram of styrene oligomers with n = 1 to 14. The peak on the extreme right originates from monomeric styrene. This is the smallest molecule (next to the molecules of the mobile phase) and is eluted last. Its elution volume is the dead volume V 0. The first small peak to the extreme left originates from excluded molecules. The corresponding volume is the interstitial volume V z (volume of liquid between the particles of the stationary phase). Between V z and V 0 the molecules with a degree of polymerisation n from 14 to 1 are eluted. The volume V 0 V z corresponds to the V o dead time n V E pore volume V p of the stationary phase (liquid which is actually in the pores). V p is the only volume useful for a separation and should therefore be as large as possible. The peak capacity depends on V p. As can be seen, the smaller the difference in molecular size (mass), the nearer the peaks are to each other. If one plots the logarithm of the molecular weight as a function of the elution volume, the resultant graph which characterises the column is in the ideal case a straight line. The calibration graph can however also be curved (see e.g. page 126). If one wants to characterise an unknown fraction or determine the molecular weight distribution of a polymer compound, the chromatogram of the sample is compared with the calibration curves. Since the elution volume corresponds to the size of a molecule and not directly to its weight (mass), this comparison is only correct for molecules of the same type (e. g. for homologues or as in the above-mentioned case polystyrenes) in the same solvent. Molecules of other types (e. g. polyamide instead of polystyrene) can, for a definite elution volume (i.e. same size) have a different density than the calibration molecules. There are conversion factors from the mostly used polystyrene standards to other substances, however, it is preferable to calibrate the column with a standard with characteristics as similar to the test sample as possible, when molecular weights are to be determined. One and the same molecule can have different sizes in different solvents (whether real or apparent) and is thereby eluted at different volumes. A coiled molecule can swell or shrink. Also a molecule can solvatise in one solvent, and thus appear larger than it really is, while it does not solvatise in another solvent. Gel chromatographic determinations of molecular weights and molecular weight distributions are simple and fast. Since they depend entirely on the elution volume, this parameter must be measured with great accuracy. The elution volume is related to the logarithm of the molar mass, and therefore even small mistakes in the determination of V E have a considerable impact on the result. One needs then a very precise and reproducibly working pump and a thermostatically controlled system or a continuous volume measurement system. Columns in series. When a separation of test samples is insufficient, it can be improved by connecting two or more columns of the same type in series. Due to this a larger pore volume can be used. When two columns are in series, however, you have not only doubled the elution volumes, but also doubled the analysis times. Should the sample contain molecules with a weight distribution exceeding the separation range of the column to be used, then two or more columns with different exclusion limits can be connected in series. In this way it is possible to separate a wide range of molecular weights in one operation. If the number of components is not too large, but still with a wide range of molecular weights, we recommend a mixed gel column with resins of different exclusion limits which enables separation of a wide range of molecular weights from 10 3 to

3 Products for GPC Products for GPC Columns for gel permeation chromatography (GPC) Phase systems. Mobile and stationary phases for SEC must meet three requirements: The mobile phase must be a good solvent for the sample, however, it should not chemically alter the sample. The sample should not show any interactions with the stationary phase. The mobile phase must not damage the stationary phase. If these three requirements are met, size exclusion chromatography is a relatively uncomplicated, convenient, versatile and fast separation technique. The solvent selected as mobile phase has to be a good solvent for the sample, however, it also has to be compatible with the stationary phase. If you are in doubt, ask our technical support for information about this point. If the sample is only partially soluble in the mobile phase, this can result in undesirable interactions between sample and stationary phase. Interactions can cause tailing, and more so delayed elution. Should a component appear after V 0, it has been somehow retained in the stationary phase. NUCLEOGEL columns for GPC The NUCLEOGEL column family comprises HPLC polymer-based columns for organic and aqueous gel permeation chromatography (GPC), gel filtration, reversed phase chromatography and ion chromatography. This chapter describes the NUCLEOGEL columns for GPC; for a detailed description of our NUCLEOGEL columns for gel filtration, reversed phase and ion chromatography please see the chapters Columns for the separation of inorganic anions (p. 78), Columns for biochemical separations (from p. 93), and Columns for food analysis (from p. 118). NUCLEOGEL GPC columns for non-aqueous eluents NUCLEOGEL GPC columns are packed with a highly cross-linked macroporous, spherical polystyrene divinylbenzene polymers matrix (PS/DVB). This material offers several advantages for size exclusion chromatography: very stable polymer with good pressure stability minimum expansion or shrinking with changing eluent polarity well suited for operation at elevated temperatures Column type Exclusion limit [kdalton] Application Column 300 x 7.7 mm 5 µm particles 10 µm particles NUCLEOGEL GPC for the separation of water-insoluble substances gel matrix polystyrene divinylbenzene, eluent in column toluene Valco type analytical columns NUCLEOGEL GPC 50 2 low molecular weight organics NUCLEOGEL GPC oligomers, oils NUCLEOGEL GPC low molecular weight polymers NUCLEOGEL GPC low molecular weight polymers NUCLEOGEL GPC polymers up to 500 kdalton NUCLEOGEL GPC molecular weight distribution of polymers NUCLEOGEL GPC Mixed gel columns: NUCLEOGEL GPC LM NUCLEOGEL GPC M NUCLEOGEL GPC M guard column 50 x 7.7 mm Columns with 600 mm length and columns for preparative separations are available on request. 124

4 Products for GPC Products for GPC Columns for gel permeation chromatography (GPC) Due to optimisation of the cross-linking chemistry the polymer shows an outstanding mechanical stability for every available pore size, combining very good pressure stability with pore volume as large as possible. The rigid structure of the PS/DVB polymer guarantees minimum expansion or shrinking of the particles when changing from polar to nonpolar organic eluents or vice versa. Thus, rapid changes of solvent polarity or temperature are possible without loss of efficiency. Mixed eluents can be used without any problems. For this reason it is no longer necessary to offer columns packed with different eluents. However, the flushing conditions recommended for certain eluent changes should be observed (s. below) Plate height as a function of flow rate column: NUCLEOGEL GPC M-10 1/N 10 5 Polystyrene Dalton Polystyrene Dalton Polystyrene Dalton Phenylhexane ml/min Procedures for changing the eluent in a NUCLEOGEL GPC column NUCLEOGEL GPC columns are packed and tested with THF, but supplied with toluene as in-column eluent. Eluent in column is toluene Change for: Change for: Change for: Change for: Hexane Perfluoroalkanes Dichloromethane Methyl ethyl ketone Ethyl acetate Chloroform Dichloroethane Cyclohexane Dimethylacetamide Pyridine Dimethylformamide Dimethylsulphoxide N-Methylpyrrolidone o-dichlorobenzene Trichlorobenzene o-chlorophenol m-cresol flush column with 2 column volumes acetone at 0.5 ml/min flush column with 2 column volumes acetone at 0.5 ml/min flush column with 2 column volumes acetone at 0.5 ml/min increase column temperature to 50 C flush column with 2 column volumes of the required eluent at 0.5 ml/min flush column with 2 column volumes of the required eluent at 0.2 ml/min and ambient temperature flush column with 2 column volumes of the required eluent at 0.2 ml/min and ambient temperature flush column with 2 column volumes of the required eluent at 0.1 ml/min increase column temperature to 60 C with 1 C/min increase column temperature to 80 C with 1 C/min increase column temperature with 1 C/min to C depending on requirements Now the column can be operated with the new eluent at the required flow. Now the column can be operated with the new eluent at a maximum of 60 C and the required flow. Now the column can be operated with the new eluent at a maximum of 80 C and the required flow. Now the column can be operated with the new eluent at the required flow. 125

5 Products for GPC Products for GPC Columns for gel permeation chromatography (GPC) polystyrene molecular weight [Dalton] NUCLEOGEL GPC M GPC often requires separation at elevated temperatures. Increasing the temperature results in an acceleration of the molecular diffusion and hence an improved GPC separation. This is especially true for viscous eluents such as dimethylformamide (DMF) or N-methylpyrrolidone (NMP) and for high molecular weight samples. Furthermore, semi-crystalline polymers, e.g. polyolefins, require elevated temperatures for dissolution and for avoiding precipitation during the separation. NUCLEOGEL GPC columns are well suited for use at elevated temperatures, because the rigid polymer matrix will tolerate numerous heating and cooling cycles without losing its separation efficiency. NUCLEOGEL GPC columns are available with 5 and 10 µm polymer beads and in many pore sizes. The diagram on the left shows calibration curves for polystyrene standards on NUCLEOGEL GPC columns with different pore sizes elution volume [ml/30 cm] Application of NUCLEOGEL GPC columns NUCLEOGEL GPC column selection for typical fields of application separation polymers with very high molecular weights (MW) polymers with high MW polymers, resins MW < polymers with low MW oils, oligomers org. compounds, MW < recommended NUCLEOGEL GPC column GPC GPC GPC x GPC M-10 / 3 x GPC M-10 2 x GPC M-5 GPC GPC x GPC x GPC GPC GPC columns 2 or 3 columns polystyrene molecular weight (dalton) For typical fields of application the column combinations shown in the diagram above have proven useful. For ordering information of NUCLEOGEL GPC columns please see page 124. For molecular weight distributions of polymers up to 5 million dalton we recommend the mixed gel columns NUCLEO- GEL GPC M-5, for higher molecular weights mixed gel columns with 10 µm particle size or even better a combination of 10 µm columns NUCLEOGEL GPC 106, 104 and 500 should be preferred. Condensation polymers and resins up to dalton can be analysed using a combination of the 5 µm NUCLEOGEL GPC columns with 10 4 Å and 500 Å pore sizes. For increased resolution, two columns each of both pore sizes can be connected in series. Low molecular weight resins, prepolymers, oils and additives require a high resolution. NUCLEOGEL GPC columns with 10 3 Å, 500 Å and 100 Å meet these requirements with their typical efficiency of theoretical plates per meter. Working ranges for polystyrene NUCLEOGEL GPC Mix [MW in dalton] 126

6 Columns for gel permeation chromatography (GPC) Application of NUCLEOGEL GPC columns Products for GPC Products for GPC GPC of dialkyl phthalates Column: VA 300/7.7 NUCLEOGEL GPC 50-5, 300 x 7.7 mm, Cat. No Eluent: Tetrahydrofuran 4 Flow rate: 1 ml/min Detection: RI 2 3 Peaks (150 µg each, injection volume 20 µl): 1. Dioctyl phthalate 1 2. Di-n-butyl phthalate 3. Diethyl phthalate 4. Dimethyl phthalate 5. Toluene 5 GPC analysis of a polyester polyol Columns: 2 x VA 300/7.7 NUCLEOGEL GPC 500-5, 300 x 7.7 mm each, Cat. No Eluent: Tetrahydrofuran Flow rate: 1 ml/min Detection: RI min min Analysis of plasticisers Column: 2 x VA 300/7.7 NUCLEOGEL GPC 100-5, 300 x 7.7 mm each, Cat. No Eluent: Tetrahydrofuran 4 3 Flow rate: 1 ml/min 2 Detection: RI Peaks: 1. Toluene 2. Dimethyl phthalate 3. Diethyl phthalate 4. Dibutyl phthalate 5. Dioctyl phthalate 6. excluded polymer 6 1 Analysis of a melamine resin Columns: 2 x 300/7.7 NUCLEOGEL GPC 100-5, 300 x 7.7 mm each, Cat.No Eluent: N,N-Dimethylformamide Flow rate: 1 ml/min Temperature: 80 C Detection: RI min min 127

7 Products for GPC Products for GPC Columns for gel permeation chromatography (GPC) Application of NUCLEOGEL GPC columns GPC separation of polystyrene standards Column: VA 300/7.7 NUCLEOGEL GPC 104-5, 300 x 7.7 mm, Cat. No Eluent: Tetrahydrofuran Flow rate: 0.5 ml/min Detection: RI Polystyrene standards [dalton] Phenylhexane (162) 8. o-dichlorobenzene 8 Analysis of polystyrene 500 Columns: 4 x VA 300/7.7 NUCLEOGEL GPC 100-5, 300 x 7.7 mm each, Cat. No Eluent: Tetrahydrofuran Detection: RI min min Separation of polystyrene standards on a mixed gel column Column: VA 300/7.7 NUCLEOGEL GPC M-5, 300 x 7.7 mm, Cat. No Eluent: Tetrahydrofuran 7 Flow rate: 0.5 ml/min Detection: RI 4 Polystyrene standards [dalton] Phenylhexane (162) 1 11 GPC analysis of polymethylmethacrylate standards (PMMA) On NUCLEOGEL columns, PMMA is easily analysed in methyl ethyl ketone (MEK), which has a lower refractive index than tetrahydrofuran and is thus especially suited for RI detection. Columns: 2 x VA 300/7.7 NUCLEOGEL GPC M-10, 300 x 7.7 mm each, Cat. No Eluent: MEK Flow rate: 1 ml/min Detection: RI PMMA standards [dalton] min min

8 Columns for gel permeation chromatography (GPC) Application of NUCLEOGEL GPC columns Products for GPC Products for GPC Analysis of pullulan polysaccharides in DMSO Due to the high viscosity of the dimethylsulphoxide elevated temperatures are required for the separation. Columns: 2 x VA 300/7.7 NUCLEOGEL GPC , Cat. No GPC , Cat. No , 300 x 7.7 mm ID each Eluent: DMSO Temperature: 70 C Flow rate: 1 ml/min Detection: RI 3 Pullulan polysaccharide standards [dalton] Separation of linear hydrocarbon standards Columns: 2 x VA 300/7.7 NUCLEOGEL GPC 50-5, 300 x 7.7 mm each, Cat. No Eluent: Tetrahydrofuran Flow rate: 1 ml/min Detection: RI Linear hydrocarbons (0.5% each) 1. C 32 H 66 MW = 450 dalton 2. C 22 H 46 MW = 310 dalton 3. C 16 H 34 MW = 226 dalton 4. C 12 H 26 MW = 170 dalton min 10 min 15 Different peak heights are due to decreasing refractive indices of the samples (n d = 1,4550 for C 32 H 66 to n d = 1,4216 for C 12 H 26 ). Analysis of stearins GPC can be used for the identification of stable intermediates produced during degradation of natural oils by microorganisms. Column: VA 300/7.7 NUCLEOGEL GPC 50-5, 300 x 7.7 mm, Cat. No Eluent: Tetrahydrofuran Flow rate: 0.5 ml/min 1 Detection: RI Peaks: 4 1. Stearic acid 2. Glyceryl monostearate 3. Glyceryl distearate 4. Glyceryl tristearate 2 3 Analysis of silicone Columns: 2 x VA 300/7.7 NUCLEOGEL GPC 500-5, 300 x 7.7 mm each, Cat. No Eluent: Toluene Flow rate: 1 ml/min Detection: RI min 9 18 min For ordering information of NUCLEOGEL GPC columns please see page

9 Products for GPC Products for GPC Columns for gel permeation chromatography (GPC) NUCLEOGEL aqua-oh columns for GPC of water-soluble compounds Column type Pore size [Å] Exclusion limit [kdalton] NUCLEOGEL aqua-oh for aqueous eluents Cat. No. Column 300 x 7.7 mm Guard column 50 x 7.7 mm hydrophilic gel matrix, particle size 8 µm; eluent in column H 2 O % NaN 3 Valco type analytical columns NUCLEOGEL aqua-oh NUCLEOGEL aqua-oh NUCLEOGEL aqua-oh Preparative NUCLEOGEL aqua-oh columns are available on request. Technical data working range [dalton] for polyethylene oxide/polyethylene glycol NUCLEOGEL aqua-oh NUCLEOGEL aqua-oh NUCLEOGEL aqua-oh ph working range 2 12 NUCLEOGEL aqua-oh columns for the GPC of water soluble samples are packed with a rigid, macroporous polymer with hydrophilic surface. The different available pore sizes allow separations of substances up to very high molecular weights. Applications: Separation of polyethylene oxide and polyethylene glycol standards Column: VA 300/7.7 NUCLEOGEL aqua-oh 50-8, 300 x 7.7 mm, Cat. No Eluent: H 2 O Flow rate: 1 ml/min Temperature: RT Detection: RI 1. PEO PEG PEG Analysis of polystyrene sulphonate standards Columns: 2 x VA 300/7.7 NUCLEOGEL aqua-oh 40-8, 300 x 7.7 mm ID each, Cat. No Eluent: 0.01 M NaH 2 PO 4, 0.2 M NaCl, ph 9 Flow rate: 1 ml/min Detection: UV, 254 nm Polystyrene sulphonate 1 standards [dalton]: PEO PEG PEG min min 130

10 Columns for gel permeation chromatography (GPC) Products for GPC Products for GPC Columns: 2 x VA 300/7.7 NUCLEOGEL aqua-oh 40-8, 300 x 7,7 mm ID each, Cat. No Eluent: 0.25 M NaNO 3, 0.01 M NaH 2 PO 4, ph 7 Flow rate: 1 ml/min Detection: RI Chromatogram of a commercial polysaccharide which contains a small amount of monosaccharide Polysaccharide analysis The following figure shows the molecular weight distribution of the polysaccharide sample. Pullulan polysaccharides were used as calibration substances. dw/dlog M 0.8 molecular weights [Dalton]: M p = M n = M w = M z = M w /M n = 1,53 Cum Ht% min log M 0 Polymer standards for GPC Polymer standards for organic eluents Polystyrene Polystyrene (PS) is prepared by anionic polymerisation. It is a very stable polymer with long shelf life. C 4 H 9 CH 2 CH H CH 2 CH Phenylhexane n Polystyrene Calibration kits Polymers are chosen to give approximately equal logarithmic molecular weight intervals. Size exclusion chromatography (SEC) or gel permeation chromatography (GPC) separates molecules according to size. If one wants to know, which elution volume V E corresponds to a given molecular size, the column has to be calibrated with a test mixture of known molecular weight distribution. Such test mixtures, called polymer standards, consist of defined fractions of specially prepared polymers. Designation Pack of M w /M n Cat. No. Polystyrene (PS) Single standards PS g phenylhexane PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g PS g

11 Products for GPC Products for GPC Polymer standards for GPC Polymer standards for organic eluents 0.5 g PS each with low molecular mass: dalton 0.5 g PS each with medium molecular mass: kdalton 0.5 g PS each with high molecular mass: kdalton Polymethylmethacrylate PMMA is prepared by anionic polymerisation at low temperature in polar solvent. CH 3 CH 2 C C O CH 3 O n Designation Pack of M w /M n Cat. No. Polystyrene calibration kits PS kit S-L x 0.5 g PS kit S-M x 0.5 g PS kit S-H x 0.5 g Polymethylmethacrylate (PMMA) Polymethylmethacrylate calibration kits PMMA kit M-L x 0.5 g g PMMA each of ,5 3, kdalton PMMA kit M-M x 0.5 g g PMMA each of kdalton Polymer standards for aqueous eluents Polyethylene oxide Water / DMF soluble polymers, recommended for calibrations in DMF. Calibration kit PEO-10 consists of 0.2 g each of PEO kdalton Polyethylene glycol Water / DMF soluble polymers, like PEO recommended for calibrations in DMF. HO CH 2 CH 2 O n CH 2 CH 2 O H n Designation Pack of M w /M n Cat. No. Polyethylene oxide (PEO) Single standards PEO g PEO g PEO g PEO g PEO g PEO g PEO g PEO g PEO g PEO g Polyethylene oxide calibration kit Kit PEO x 0.2 g Polyethylene glycol (PEG) Single standards Di-EG g Dimer Tetra-EG g Tetramer PEG g PEG g PEG g PEG g PEG g PEG g PEG g

12 Products for GPC Products for GPC Polymer standards for GPC Polymer standards for aqueous eluents Calibration kit PEG-10 consists of 10 x 0.5 g PEG as follows dalton Polysaccharides The SHODEX polysaccharide calibration kit P-82 consists of unbranched, linear polysaccharides with the following structure, also known as pullulan: Designation Pack of M w /M n Cat. No. PEG g PEG g Polyethylene glycol calibration kit Kit PEG x 0.5 g Polysaccharides Polysaccharide calibration kit Kit P-82 (Pullulan) 8 x 0.2 g H O OH CH 2 H OH H O H OH H O H CH 2 OH O H OH H H The polysaccharides are easily soluble in water and their solutions are very stable when stored properly. The calibration kit P-82 consists of 0.2 g each of the polysaccharides listed in the table. OH H O H CH 2 OH O H OH H H OH H n Conditions for the use of polymer standards Stability Polymers such as polystyrene or polymethylmethacrylate are very stable under normal operating conditions. Polystyrene at very high temperatures (> 140 C) is more susceptible to degradation leading to incorrect molecular weights or broad molecular weight distributions. Molecular weight Polymers of high molecular weight (> ) are more likely to suffer from mechanical degradation during dissolution. Sample should be subjected to gently shaking or swirling by hand, and left overnight to swell and dissolve slowly. Concentration This should be kept as low as possible consistent with the available detection limits. A recommended value is 0.2 % (w/v) for low and medium molecular weights when a refractive index is used, but for molecular weights above the concentration should be reduced to 0.05%, and even to 0.02% for molecular weights above If a UV detector is used, the concentration may be reduced by a further factor of 10. Overloading If the concentration is too high, or if the injection volume too large, then overloading may occur, particularly with high molecular weights. The effect on the molecular weight distribution may be seen as severe broadening, tailing, bimodality or lowering of the observed molecular weights. Lowering the concentration or the injection volume or both will correct the problem. Injection volumes of 20 to 50 µl are recommended. Polymers other than polystyrene should be stored at or under +5 C under nitrogen. Handling of polysaccharide standards Preparation of calibration solutions The following procedure is recommended for preparation of a standard solution of any component of the calibration kit: 1) Weigh 0.5 to 1 mg of the polysaccharide in a bottle 2) Add distilled water to prepare a % solution and let it stand for 12 to 24 h at a maximum of 25 C. Do not stir the solution before the particles have swelled completely. Warming of the solution will cause decomposition as well. 3) After confirming that the agglomerate particles have swelled completely, stir the water gently until a homogeneous mixture is obtained. 4) Finally, filter the solution with a filter of 0.45 µm pore size. 5) Prepared calibration solutions should be stored in a refrigerator to avoid decomposition by microorganisms or enzymes. For storage, the ph value of the solution should be between 5 and % sodium azide can be added as an antiseptic. In the solid state the polysaccharides of the calibration kit are very stable, if they are stored dry and below 25 C. If humidity is high, we recommend storing the standards in a desiccator. For detection of polysaccharides only an Rl detector can be used, UV detection is not possible! 133

13 NUCLEODUR spherical silicas In principle preparative HPLC follows the same rules as analytical scale chromatography. However, there are important differences in the aims of the two techniques. In analytical HPLC chromatographers focus on peak shape, and resolution of all eluted analytes, whereas in preparative chromatography yield and purity of the final product, as well as cost-effectiveness of the method, are emphasized. NEW throughput Stability Physical stability: This totally spherical, 100% synthetic silica gel exhibits an outstanding mechanical stability, even at high pressures, and elevated flow rates of the mobile phase. Moreover, the material is re-usable. After several cycles of repeated packing, no significant changes in pressure drop can be observed. This is an important prerequisite for process-scale applications. back pressure [bar] Packing # demands on preparative separations Chemical stability: The utmost purity of the base silica and the exceptional silane bonding chemistry minimizes the risk of dissolution, or hydrolysis at ph extremes. The following chromatograms show the retention behaviour at ph values of 1.5 and 10.0 for NUCLEODUR C 18 endcapped. yield Requirements for a high performance silica Purity For achieving an optimum resolution and symmetric peak shapes, a highly pure silica is required. It is well known, that metal ions, on the surface of silica largely are responsible for unwanted interactions with ionizable analytes, e. g. amines or phenolic compounds. The unique manufacturing process of NUCLEODUR provides a highly pure silica virtually free of metal impurities, and low acidic surface silanols. The following table shows the elementary analysis (metal ions), measured by AAS, of NUCLEODUR silica, 100 Å, 5 µm. Aluminium < 5 ppm Iron < 5 ppm Sodium < 5 ppm Calcium < 10 ppm Titanium < 1 ppm Zirconium < 1 ppm Arsenic < 0.5 ppm Mercury < 0.05 ppm purity Separation of uracil, veratrol, toluene and ethylbenzene at ph 1.5 Column: CC 30/3 NUCLEODUR C 18 ec Mobile phase: ACN/H 2 O (65:35, v/v), TFA, ph 1.5 Flow rate: 1.0 ml/min Temperature: 25 C Detection: UV, 254 nm min Stability of NUCLEODUR at ph 1.5 over 1000 cycles (conditions see chromatogram) inj. 134

14 NUCLEODUR spherical silicas Separation of theophylline and caffeine at ph 10 Column: CC 30/3 NUCLEODUR C 18 ec Mobile phase: mau MeOH/aq. NH 3 (20:80,v/v), ph Flow rate: 0.5 ml/min Temperature: 25 C Detection: UV, 254 nm inj. 750 inj. 500 inj. 250 inj. Start min Loadability Loadability, probably the most important feature for preparative LC applications, is determined by pore size, pore volume and surface area of the packing. However, it can also be influenced by the molecular weight of the analytes. In figure 8 the mass loading curve for acetophenone and butyrophenone on a NUCLEODUR C 18 ec column describes the correlation between the increase of column loading and the decrease of separation efficiency. The following chromatograms show the separation of a phthalate mixture on an analytical NUCLEODUR column, compared with the same separation under mass-overload conditions on a semipreparative column. However, without suitable selectivity of the stationary phase, and a sufficient solubility of the compounds in the mobile phase, a good preparative separation in high yields cannot be achieved. Surface bonding chemistry The efficiency of a separation is controlled by particle size and selectivity of the stationary phase. NUCLEODUR is available in 7 different particle sizes (5, 10, 12, 16, 20, 30 and 50 µm) as silica and with RP derivatization (C 8 and C 18 ). The exceptional surface coverage of monomerically bonded alkylsilanes combined with an exhaustive endcapping, results in a surface with lowest silanol activity. The elution of critical compounds such as basic drugs is possible without adsorption or peak deformation. The wide range of different particle sizes allow the use in analytical, preparative HPLC and for scale-up applications. Separation of phenols Column: EC 250/4 NUCLEODUR C18 ec Mobile phase: Methanol/H 2 O, 60:40 (v/v) Flow: 1.0 ml/min Injection volume: 4 µl Temperature: 30 C Detection: UV 254nm Peaks: 1) Pyrocatechol 2) Resorcinol 1 3 3) Phenol 4 2 4) Guaiacol 5) Veratrole min Loading curve Column: EC 250/4.6 NUCLEODUR C 18 ec Mobile phase: acetonitrile/h 2 O 80:20 (v/v) Flow: 1.0 ml/min Temperature: 25 C acetophenone Detection: UV nm butyrophenone theoret. plates / m µg / compound For ordering information of NUCLEODUR packings see pages 137 and

15 Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied. The high standard of quality, the broad range of available packing materials, and ready-to-use columns, combined with efficient instrumentation, allow short analysis times optimum selectivity high sensitivity of detection These parameters can be optimised by variation of particle size and pore structure modification of the packing column dimension eluent and chromatographic conditions We offer a versatile programme of HPLC packings, briefly summarised in the following groups: silica, spherical particles totally porous available under the trade names NUCLEOSIL and NUCLEODUR silica, irregular particles totally porous available under the trade names POLYGOSIL and POLYGOPREP NUCLEOPREP spherical silicas 12 µm modified as well as unmodified are still available on request. All silica packing materials are available as unmodified silica, as well as with chemically bonded phases with different organic groups and thus specific selective behaviour. At this point it should be mentioned, that in practice most separation problems can be solved using only a few stationary phases, if the different selectivities of eluents are taken into account. However, in order to optimize certain separations, advantage can be taken of the specific selectivities of chemically bonded phases, which can save time and effort needed for determining the best separation conditions. The actual particle size of the packing plays an important role for the efficiency of a column. In general, higher efficiencies are achieved with smaller particles. Depending on the separation problem, however, somewhat larger particles (e. g. 7 µm instead of 5 µm) can still give good efficiency with shorter analysis times and longer column life. Consistent use of very narrow particle size distributions is another means of optimising separations. We supply packings with very narrow particle size distribution for optimum analytical performance. Furthermore, the permeability and the pressure required for a given eluent velocity depend strongly on the particle size and distribution. silica packings As silica packings for HPLC MACHEREY-NAGEL supply spherical NUCLEOSIL and NUCLEODUR as well as irregular POLYGOSIL and ( 12 µm) POLYGOPREP. These types are available as unmodified silica as well as with chemically bonded phases. For a detailed description of our high performance silica NUCLEOSIL and columns packed with these phases please see pages All available modifications are listed on page 18. The high performance sillica NUCLEODUR is described on page 134. For POLYGOSIL and POLYGOPREP packings see page 148 and following. 136

16 NUCLEOSIL and NUCLEODUR spherical silicas Octadecyl phases -(CH 2 ) 17 CH 3 C 18 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of nonpolar to moderately polar compounds such as fatty acids glycerides polycyclic aromatics esters (phthalates) fat-soluble vitamins steroids prostaglandins PTH amino acids etc. NUCLEOSIL 50-5 C 18 ec spherical silica, octadecyl phase, endcapped (HMDS), 14.5% C, pore size 50 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEOSIL C 18 AB spherical silica, octadecyl phase, endcapped, base deactivated, polymer coating, 24% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEODUR 100 C 18 ec spherical silica, octadecyl phase, endcapped (HMDS), 17.5% C, pore size ~110 Å, ph stability at 20 C: 1 9 NEW NUCLEODUR C 18 NUCLEODUR C 18 NUCLEODUR C 18 NUCLEODUR C 18 NUCLEODUR C 18 particle size 10 µm particle size 16 µm particle size 20 µm particle size 30 µm particle size 50 µm pack of 100 g pack of 1000 g NUCLEOSIL 100 C 18 spherical silica, octadecyl phase, endcapped (HMDS), 15% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL NUCLEOSIL NUCLEOSIL NUCLEOSIL C C C C 18 particle size 3.5 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 120 C 18 spherical silica, octadecyl phase, endcapped (HMDS), 11% C, pore size 120 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 18 NUCLEOSIL C 18 NUCLEOSIL C 18 NUCLEOSIL C 18 particle size 3.5 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g

17 NUCLEOSIL spherical silicas Wide pore silica packings Silica as HPLC packing has several advantages: defined pore size and surface mechanical stability of the silica structure numerous methods for surface modification are available Many biologically interesting molecules can not be separated using conventional narrow pore silicas with pore diameters of about 100 Å. This is why we offer a complete line of wide pore packings with pore sizes of 300, 500, 1000 and 4000 Å. These materials can also be used for size exclusion chromatography (SEC). NUCLEOSIL 300 C 18 spherical silica, octadecyl phase, endcapped (HMDS), 6.5% C, pore size 300 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 18 NUCLEOSIL C 18 NUCLEOSIL C 18 particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL C 18 spherical silica, octadecyl phase, endcapped (HMDS), 2% C, pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 18 spherical silica, octadecyl phase, endcapped (HMDS), ~ 1% C, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 18 spherical silica, octadecyl phase, endcapped (HMDS), <1% C, pore size 4000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

18 NUCLEOSIL spherical silicas Octyl phases -(CH 2 ) 7 CH 3 C 8 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of moderately to highly polar (water-soluble) compounds such as steroids nucleosides cyclodextrins pharmacological plant constituents NUCLEOSIL 50-5 C 8 ec spherical silica, octyl phase, endcapped (HMDS), 9% C, pore size 50 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEOSIL C 8 ec spherical silica, octyl phase, endcapped (HMDS), 9% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEOSIL 100 C 8 spherical silica, octyl phase, not endcapped, 8.5% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 8 NUCLEOSIL C 8 NUCLEOSIL C 8 particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 120 C 8 spherical silica, octyl phase, not endcapped, 6.5% C, pore size 120 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 8 NUCLEOSIL C 8 NUCLEOSIL C 8 NUCLEOSIL C 8 particle size 3.5 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 300 C 8 spherical silica, octyl phase, not endcapped, ~ 3% C, pore size 300 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 8 NUCLEOSIL C 8 NUCLEOSIL C 8 particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL C 8 spherical silica, octyl phase, not endcapped, <1% C, pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

19 NUCLEOSIL spherical silicas Phenyl phases -(CH 2 ) 3 C 6 H 5 phases are suited for reversed phase chromatography and ion pairing chromatography for separation of moderately polar compounds. The retention characteristics are similar to the C 8 phase, but with different selectivity for polycyclic aromatic hydrocarbons, polar aromatics, fatty acids etc. NUCLEOSIL C 6 H 5 ec spherical silica, phenyl phase, endcapped (HMDS), 8% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEOSIL 100 C 6 H 5 spherical silica, phenyl phase, not endcapped, 8% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 6 H 5 NUCLEOSIL C 6 H 5 particle size 5 µm particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 6 H 5 spherical silica, phenyl phase, not endcapped, 6,5% C, pore size 120 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 6 H 5 spherical silica, phenyl phase, not endcapped, ~ 3% C, pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 6 H 5 spherical silica, phenyl phase, not endcapped, pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 6 H 5 spherical silica, phenyl phase, not endcapped, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

20 NUCLEOSIL spherical silicas Butyl phases -(CH 2 ) 3 CH 3 C 4 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of macromolecules and hydrophobic substances. Retention times on butyl phases are shorter than on C 8 and C 18 phases. NUCLEOSIL C 4 spherical silica, butyl phase, not endcapped, pore size 120 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g NUCLEOSIL 300 C 4 spherical silica, butyl phase, endcapped (HMDS), ~ 2% C, pore size 300 Å, ph stability at 20 C: 1 9 NUCLEOSIL C 4 NUCLEOSIL C 4 NUCLEOSIL C 4 particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 4000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

21 NUCLEOSIL spherical silicas Dimethyl phases -(CH 3 ) 2 Dimethyl phases are suited for reversed phase chromatography and ion-pairing chromatography. Retention times on the dimethyl phase are much shorter than for the other RP phases. NUCLEOSIL C 2 spherical silica, dimethyl phase, not endcapped, 3.5% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g Cyano phases (nitrile) CN phases are suited for reversed phase chromatography and for normal phase chromatography: Normal phase chromatography: with low-polarity solvents for many compounds, which can also be separated on unmodified silica, however, due to the -(CH 2 ) 3 CN rapid equilibration much more suitable for gradient separations Reversed phase chromatography: with different selectivity than C 18, C 8 or phenyl modified packings. NUCLEOSIL 100 CN spherical silica, cyano phase (nitrile), 5% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL CN NUCLEOSIL CN particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL CN spherical silica, cyano phase (nitrile), pore size 120 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL CN spherical silica, cyano phase (nitrile), pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL CN spherical silica, cyano phase (nitrile), pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

22 NUCLEOSIL spherical silicas Nitro phases -(CH 2 ) 3 NO 2 NO 2 phases are suited for the separation of compounds with double bonds or for aromatic compounds. NUCLEOSIL 100 NO 2 spherical silica, nitro phase, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL NO 2 NUCLEOSIL NO 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g Diol phases OH phases are suited for reversed phase chromatography and normal phase chromatography; they are less polar than unmodified silica and very easily wettable with water. -(CH 2 ) 3 O CH 2 CH CH 2 OH OH NUCLEOSIL OH (Diol) spherical silica, diol phase, 5% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL OH (Diol) spherical silica, diol phase, pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL OH (Diol) spherical silica, diol phase, pore size 500 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL OH (Diol) spherical silica, diol phase, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL OH (Diol) spherical silica, diol phase, pore size 4000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g

23 NUCLEOSIL and NUCLEODUR spherical silicas Unmodified silica Due to its narrow pore structure, NUCLEOSIL 50 is recommended for adsorption chromatography, while NUCLEOSIL and NUCLEODUR 100 as well as NUCLEOSIL 120 can be used for adsorption as well as for partition chromatography. SiOH NUCLEOSIL 50 spherical silica, unmodified, pore size 50 Å, pore volume 0.8 ml/g, surface (BET) 420 m 2 /g, density 0.45 g/ml, pressure stability 600 bar NUCLEOSIL 50-3 NUCLEOSIL 50-5 NUCLEOSIL 50-7 NUCLEOSIL particle size 3 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 100 spherical silica, unmodified, pore size 100 Å, pore volume 1 ml/g, surface (BET) 350 m 2 /g, density 0.36 g/ml, pressure stability 600 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 3 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEODUR 100 spherical silica, unmodified, pore size ~110 Å, pore volume 0.9 ml/g, surface (BET) 340 m 2, density 0.47 g/ml, pressure stability 800 bar NEW NUCLEODUR NUCLEODUR NUCLEODUR NUCLEODUR NUCLEODUR particle size 10 µm particle size 16 µm particle size 20 µm particle size 30 µm particle size 50 µm pack of 100 g pack of 1000 g NUCLEOSIL 120 spherical silica, unmodified, pore size 120 Å, pore volume 0,65 ml/g, surface (BET) 200 m 2 /g, density 0.55 g/ml, pressure stability 800 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 3 µm particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 300 spherical silica, unmodified, pore size 300 Å, pore volume 0.8 ml/g, surface (BET) 100 m 2 /g, density 0.45 g/ml, pressure stability 400 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g

24 NUCLEOSIL spherical silicas NUCLEOSIL 500 spherical silica, unmodified, pore size 500 Å, pore volume 0.8 ml/g, surface (BET) 35 m 2 /g, density 0.45 g/ml, pressure stability 400 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 1000 spherical silica, unmodified, pore size 1000 Å, pore volume 0.8 ml/g, surface (BET) 25 m 2 /g, density 0.45 g/ml, pressure stability 300 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL 4000 spherical silica, unmodified, pore size 4000 Å, pore volume 0.7 ml/g, surface (BET) 10 m 2 /g, density 0.48 g/ml, pressure stability 300 bar NUCLEOSIL NUCLEOSIL NUCLEOSIL particle size 5 µm particle size 7 µm particle size 10 µm pack of 10 g pack of 100 g

25 NUCLEOSIL spherical silicas Amino phases -(CH 2 ) 3 NH 2 NH 2 phases feature versatile applicability: in normal phase chromatography with hexane, CH 2 CI 2 or isopropanol as mobile phase for polar compounds such as substituted anilines, esters, chlorinated pesticides etc. in aqueous-organic eluent systems for normal phase chromatography of polar compounds like carbohydrates as weak anion exchanger for anions and organic acids using common buffers (e. g. acetate or phosphate) in conjunction with organic modifiers (e. g. acetonitrile). NUCLEOSIL 100 NH 2 spherical silica, amino phase, 3.5% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL NH 2 NUCLEOSIL NH 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g NUCLEOSIL NH 2 spherical silica, amino phase, pore size 120 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g NUCLEOSIL NH 2 spherical silica, amino phase, pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g Dimethylamino phases -(CH 2 ) 3 N(CH 3 ) 2 The DMA phase is a weakly basic anion exchanger for the separation of many anions; it can also be used in a similar way as the NH 2 phase. NUCLEOSIL 100 N(CH 3 ) 2 spherical silica, dimethylamino phase, 4% C, pore size 100 Å, ph stability at 20 C: 1 9 NUCLEOSIL N(CH 3 ) 2 NUCLEOSIL N(CH 3 ) 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g

26 NUCLEOSIL spherical silicas Cation exchanger, strongly acidic -(CH 2 ) 3 SO 3 Na The cation exchanger NUCLEOSIL SA is used for ion exchange chromatography. It is a strongly acidic cation exchanger with sulphonic acid modification and a capacity of about 1 meq/g NUCLEOSIL 100 SA spherical silica, strongly acidic cation exchanger, sulphonic acid modification, 6.5% C, pore size 100 Å, ph stability at 20 C: NUCLEOSIL SA NUCLEOSIL SA particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g Anion exchanger, strongly basic -(CH 2 ) 3 CH 2 N + (CH 3 ) 3 Cl The anion exchanger NUCLEOSIL SB is used for ion exchange chromatography. It is a strongly basic anion exchanger with a quaternary ammonium modification and a capacity of about 1 meq/g. NUCLEOSIL 100 SB spherical silica, strongly basic anion exchanger, quaternary ammonium modification, 10% C, pore size 100 Å, ph stability at 20 C: NUCLEOSIL SB NUCLEOSIL SB particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g

27 POLYGOSIL and POLYGOPREP irregular silicas POLYGOSIL and POLYGOPREP are groups of irregular, totally porous silicas. For many applications, these materials are a cost-efficient alternative compared to spherical silica. The advantages of these packings are porous, irregular particles with controlled surface structure high efficiency due to narrow particle size distribution high reproducibility from lot to lot high load capacity and recovery rates the economical alternative for preparative and process scale chromatography POLYGOSIL and POLYGOPREP are available with mean pore diameters of 60, 100, 300 and 1000 Å. Like our spherical silicas, POLYGOSIL and POLYGOPREP too can be supplied as unmodified silica as well as with chemically bonded phases. They are also fractionated to a narrow particle size distribution. The main advantage of irregular materials compared to spherical silica is the lower price. For mainly analytical separations we offer POLYGOSIL with particle sizes of 5, 7 and 10 µm. For preparative separations POLYGOPREP packings are available with particle sizes of 12, 20, 30, 50, 80 and 130 µm. The modification of POLYGOSIL and POLYGOPREP packings follows the same processes as for spherical silica bonded phases. For analytical data and available pore sizes and modifications please refer to the following pages. 148

28 POLYGOSIL and POLYGOPREP irregular silicas Octadecyl phases -(CH 2 ) 17 CH 3 C 18 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of nonpolar to moderately polar compounds such as fatty acids glycerides polycyclic aromatics esters (phthalates) fat-soluble vitamins steroids prostaglandins PTH amino acids etc. POLYGOSIL 60 C 18 irregular silica, octadecyl phase, endcapped (HMDS), 12% C, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL POLYGOSIL POLYGOSIL 60-5 C C C 18 particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g POLYGOPREP 60 C 18 irregular silica, preparative grade, octadecyl phase, not endcapped *, 12% C, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP C C C C C C 18 particle size µm µm µm µm µm µm pack of 100 g pack of 1000 g POLYGOSIL 100 C 18 irregular silica, octadecyl phase, endcapped (HMDS), 14% C, pore size 100 Å, ph stability at 20 C: 1 9 POLYGOSIL POLYGOSIL POLYGOSIL C C C 18 particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g POLYGOPREP 100 C 18 irregular silica, preparative grade, octadecyl phase, not endcapped *, 14% C, pore size 100 Å, ph stability at 20 C: 1 9 POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP C C C C 18 particle size µm µm µm µm pack of 100 g pack of 1000 g POLYGOSIL C 18 irregular silica, octadecyl phase, endcapped (HMDS), 4% C, pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g * On request, these POLYGOPREP RP phases can be endcapped at extra cost 149

29 POLYGOSIL and POLYGOPREP irregular silicas POLYGOPREP 300 C 18 irregular silica, preparative grade, octadecyl phase, endcapped (HMDS), 4% C, pore size 300 Å, ph stability at 20 C: 1 9 POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP C C C C 18 particle size µm µm µm µm pack of 100 g pack of 1000 g POLYGOSIL C 18 irregular silica, octadecyl phase, endcapped (HMDS), ~ 1% C, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g POLYGOPREP 1000 C 18 irregular silica, preparative grade, octadecyl phase, endcapped (HMDS), ~ 1% C, pore size 1000 Å, ph stability at 20 C: 1 9 POLYGOPREP C 18 POLYGOPREP C 18 particle size µm µm pack of 100 g pack of 1000 g Octyl phases -(CH 2 ) 7 CH 3 C 8 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of moderately to highly polar (water-soluble) compounds such as steroids nucleosides cyclodextrins pharmacological plant constituents POLYGOSIL 60 C 8 irregular silica, octyl phase, not endcapped, 7% C, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL 60-5 C 8 POLYGOSIL 60-7 C 8 POLYGOSIL C 8 particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g POLYGOPREP 60 C 8 irregular silica, preparative grade, octyl phase, not endcapped *, 7% C, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOPREP C 8 POLYGOPREP C 8 POLYGOPREP C 8 POLYGOPREP C 8 particle size µm µm µm µm pack of 100 g pack of 1000 g * On request, these POLYGOPREP RP phases can be endcapped at extra cost 150

30 POLYGOSIL and POLYGOPREP irregular silicas Butyl phases -(CH 2 ) 3 CH 3 C 4 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of macromolecules and hydrophobic substances. Retention times on butyl phases are shorter than on C 8 and C 18 phases. POLYGOSIL C 4 irregular silica, butyl phase, endcapped (HMDS), ~ 1% C, pore size 300 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g POLYGOPREP 300 C 4 irregular silica, preparative grade, butyl phase, endcapped (HMDS), ~ 1% C, pore size 300 Å, ph stability at 20 C: 1 9 POLYGOPREP C 4 POLYGOPREP C 4 POLYGOPREP C 4 POLYGOPREP C 4 particle size µm particle size µm particle size µm particle size µm pack of 100 g pack of 1000 g POLYGOSIL C 4 irregular silica, butyl phase, endcapped (HMDS), < 1% C, pore size 1000 Å, ph stability at 20 C: 1 9 particle size 7 µm pack of 10 g pack of 100 g POLYGOPREP 1000 C 4 irregular silica, preparative grade, butyl phase, endcapped (HMDS), < 1% C, pore size 1000 Å, ph stability at 20 C: 1 9 POLYGOPREP C 4 POLYGOPREP C 4 particle size µm particle size µm pack of 100 g pack of 1000 g

31 POLYGOSIL and POLYGOPREP irregular silicas Cyano phases (nitrile) CN phases are suited for reversed phase chromatography and for normal phase chromatography: Normal phase chromatography: with low-polarity solvents for many compounds, which can also be separated on unmodified silica, however, due to the -(CH 2 ) 3 CN rapid equilibration much more suitable for gradient separations Reversed phase chromatography: with different selectivity than C 18, C 8 or phenyl modified packings. POLYGOSIL 60 CN irregular silica, cyano phase (nitrile), pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL 60-5 CN POLYGOSIL CN particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g POLYGOPREP 60 CN irregular silica, preparative grade, cyano phase (nitrile), pore size 60 Å, ph stability at 20 C: 1 9 POLYGOPREP CN POLYGOPREP CN POLYGOPREP CN particle size µm particle size µm particle size µm pack of 100 g pack of 1000 g Nitro phases -(CH 2 ) 3 NO 2 NO 2 phases are suited for the separation of compounds with double bonds or for aromatic compounds. POLYGOSIL 60 NO 2 irregular silica, nitro phase, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL 60-5 NO 2 POLYGOSIL NO 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g

32 POLYGOSIL and POLYGOPREP irregular silicas Unmodified silica Due to their narrow pore structure, POLYGOSIL and POLY- GOPREP 60 are recommended for adsorption chromatography, while POLYGOSIL and POLYGOPREP 100 can be used for adsorption as well as for partition chromatography. SiOH POLYGOSIL 60 irregular silica, unmodified, pore size 60 Å, pore volume 0.75 ml/g, surface (BET) 350 m 2 /g, density 0.45 g/ml, pressure stability 600 bar POLYGOSIL 60-5 POLYGOSIL 60-7 POLYGOSIL particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g POLYGOPREP 60 irregular silica, unmodified, preparative grade, pore size 60 Å, pore volume 0.75 ml/g, surface (BET) 370 m 2 /g, density 0.45 g/ml, pressure stability 600 bar POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP particle size µm µm µm µm µm µm pack of 1000 g pack of 5000 g POLYGOSIL 100 irregular silica, unmodified, pore size 100 Å, pore volume 1 ml/g, surface (BET) 280 m 2 /g, density 0.35 g/ml, pressure stability 400 bar POLYGOSIL POLYGOSIL POLYGOSIL particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g POLYGOPREP 100 irregular silica, unmodified, preparative grade, pore size 100 Å, pore volume 1 ml/g, surface (BET) 280 m 2 /g, density 0.35 g/ml, pressure stability 400 bar POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP particle size µm µm µm µm µm µm pack of 1000 g pack of 5000 g

33 POLYGOSIL and POLYGOPREP irregular silicas POLYGOSIL irregular silica, unmodified, pore size 300 Å, pore volume 0.8 ml/g, surface (BET) 100 m 2 /g, density 0.45 g/ml, pressure stability 400 bar particle size 7 µm pack of 10 g pack of 100 g POLYGOPREP 300 irregular silica, unmodified, preparative grade, pore size 300 Å, pore volume 0.8 ml/g, surface (BET) 100 m 2 /g, density 0.45 g/ml, pressure stability 400 bar POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP particle size µm µm µm µm pack of 100 g pack of 1000 g POLYGOSIL irregular silica, unmodified, pore size 1000 Å, pore volume 0.8 ml/g, surface (BET) 25 m 2 /g, density 0.45 g/ml, pressure stability 300 bar particle size 7 µm pack of 10 g pack of 100 g POLYGOPREP 1000 irregular silica, unmodified, preparative grade, pore size 1000 Å, pore volume 0.8 ml/g, surface (BET) 35 m 2 /g, density 0.45 g/ml, pressure stability 300 bar POLYGOPREP POLYGOPREP POLYGOPREP POLYGOPREP particle size µm µm µm µm pack of 100 g pack of 1000 g

34 POLYGOSIL and POLYGOPREP irregular silicas Amino phases -(CH 2 ) 3 NH 2 NH 2 phases feature versatile applicability: in normal phase chromatography with hexane, CH 2 CI 2 or isopropanol as mobile phase for polar compounds such as substituted anilines, esters, chlorinated pesticides etc. in aqueous-organic eluent systems for normal phase chromatography of polar compounds like carbohydrates as weak anion exchanger for anions and organic acids using common buffers (e. g. acetate or phosphate) in conjunction with organic modifiers (e. g. acetonitrile). POLYGOSIL 60 NH 2 irregular silica, amino phase, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL 60-5 NH 2 POLYGOSIL NH 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g POLYGOPREP 60 NH 2 irregular silica, preparative grade, amino phase, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOPREP NH 2 POLYGOPREP NH 2 POLYGOPREP NH 2 particle size µm particle size µm particle size µm pack of 100 g pack of 1000 g Dimethylamino phases -(CH 2 ) 3 N(CH 3 ) 2 The DMA phase is a weakly basic anion exchanger for the separation of many anions; it can also be used in a similar way as the NH 2 phase. POLYGOSIL 60 N(CH 3 ) 2 irregular silica, dimethylamino phase, pore size 60 Å, ph stability at 20 C: 1 9 POLYGOSIL 60-5 N(CH 3 ) 2 POLYGOSIL N(CH 3 ) 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g

35 Packings for CLC Packings for CLC Adsorbents for conventional column chromatography The importance of classical low pressure column chromatography is mainly due to the following factors: Economical packings After preparation, the packings are fractionated by sieving. Generally speaking: the larger the particle, the easier, faster and cheaper classification can be performed. Also, the expense for preparation decreases with increasingly broader particle size distribution. For these reasons the prices for CLC packings are considerably lower. Low operating pressure The working pressure for a chromatographic column is inversely proportional to the square of the particle diameter of the packing employed. Columns with very coarse particle fractions or very short columns can already be operated with a low hydrostatic pressure of the eluent. Columns with small particle sizes (20 to 50 µm) require a higher hydrostatic pressure or application of a peristaltic pump in order to achieve optimum flow rates. Simple packing procedure Depending on the type of separation material and its particle size, different techniques can be used. Adsorbents which consist of compact, high density particles (e.g. aluminium oxide or silica) can be filled dry into the column. For a higher packing density it can be useful to subject the column to a vibration during packing. As an alternative, adsorbents can be packed as a slurry. For low density materials (such as polymer resins or cellulose) and for all small particles this is the method of choice to obtain even, dense packings. The ratio between column length and diameter should be about 10:1. Packing of the column should always be performed evenly, because otherwise the substances will not be eluted in compact bands but, due to formation of channels, are separated in irregular serrated bands. This can result in the simultaneous presence of different substances at the same height of the column. Columns should never run dry, i. e. the adsorbent has always to be covered with liquid. Low expense for instrumentation The substances to be separated are introduced into the column at the moment when the liquid level has just reached the top of the stationary phase. When the sample solution has completely percolated into the adsorbent, the packing can be covered with a filter (wadding, paper, a wire screen, glass fibre paper, or quartz sand) in order to avoid that the eluent stirs up the upper layer of the adsorbent. With the addition of the eluent the separation proper is started. The eluate which leaves the lower end of the column, contains in certain intervals, depending on the separation effect of the column the individual substances or classes of substances. The substances in question are determined and, if necessary, isolated from the eluate, which is normally collected in numerous small portions, with the aid of a fraction collector. All chromatographic methods with the exception of thin layer chromatography use columns for the separation process. The term column chromatography (CLC), however, stands for a classical liquid chromatographic procedure, which has been developed in the early 20th century by the Russian botanist and biochemist M.S.Tswett. In the course of time, this method has found its place in many laboratories for preparative purposes as well as for reaction control in organic syntheses. The packings used for CLC in general have a particle size between 20 and 200 µm. Requirements as to the uniformity of the particles are not very large. For products for flash chromatography see pages The following pages provide a detailed description of the adsorbents for column chromatography manufactured by MACHEREY-NAGEL. When choosing a column packing, in practice you have to consider the properties of the substances to be separated including impurities or components of no interest and then select the proper adsorbent. As a rule of thumb you can consider: for lipophilic substances aluminium oxide, silica, acetylated cellulose, polyamide for hydrophilic substances cellulose, cellulose ion exchangers, kieselguhr, polyamide In addition, it is important to consider possible reactions between the compounds and the adsorbent or eluent. In practice, silica gel is the preferred stationary phase for organic chemical separations, and only if silica is clearly not suited for a given problem, other stationary phases are used. 156

36 Packings for CLC Packings for CLC Silica adsorbents for low pressure column chromatography Standard silica Silica is the most important adsorbent for column chromatography, because it can be used for most adsorption chromatographic separation problems. Silica is a highly porous, amorphous silicic acid in the form of hard, opalescent particles, which is prepared by precipitation of water glass with sulphuric acid. Precipitation and work-up conditions determine the special properties of the silica. Specific surface area, specific pore volume, mean pore size Silica special grades Silica FIA FIA silica gels are manufactured in two grades. The FIA procedure (fluorescence indicator adsorption procedure) is used for the determination of the content of hydrocarbon groups in the testing of liquid fuels. The procedure is defined in the German standard method DIN and the US standard method ASTM D T. The FIA method determines saturated hydrocarbons, olefins and aromatic hydrocarbons of a sample chromatographically by adsorption and desorption in a column filled with FIA silica, in the presence of a fluorescent dye mixture. Silica E Silica E, particle size mm (18 35 mesh), is used for the determination of the total amount of combustible organic substances in waste gases according to Ixfeld and and others depend on the preparation conditions. Especially important for the reproducibility of individual silica batches is a careful control of the production conditions and subsequent grinding and sieving of the silica. Changes in the particle size distribution can cause considerable variations in the separation results. For higher demands on the performance of column packings we recommend our high-purity irregular POLYGOPREP silicas (see page 153). Buck (H. Ixfeld et al., Brennstoff-Chem. 47 (1966) 79 83, Brennstoff-Chem. 50 (1969) (both in German)). It is a silica specifically selected for this purpose. The procedure by Ixfeld and Buck is suited for the determination of carbon concentrations between approx. 20 and 400 mg C/Nm 3 with a relative standard deviation of 3%. Sampling and analytical determination are performed in two steps. For sampling, the organic substances are quantitatively adsorbed on silica E. For the analytical determination, the adsorbed organic substances are desorbed at elevated temperatures and burned in an oxygen stream. The carbon dioxide formed is adsorbed in Ba(OH) 2 solution and quantitated titrimetrically. Since the silica does not adsorb carbon dioxide, the CO 2 always present in waste gases is already removed during sampling and does not interfere with the analysis of the combustible substances. Designation Particle size 1 kg 5 kg 25 kg Silica standard grades Silica 60, pore size ~ 60 Å; pore volume ~ 0.75 ml/g; spec. surface BET ~ 450 m 2 /g Silica 60, mm Silica 60, mm Silica 60, mm Silica 60, mm mesh Silica 60 M, mm mesh Silica 60, mm mesh Silica 60, mm mesh Silica 60, mm mesh Silica 60, < mm +230 mesh Silica 60, < 0.08 mm +190 mesh Silica 60, mm mesh Silica 60, mm mesh Silica 60, mm mesh Silica FIA for the fluorescence indicator adsorption procedure for the determination of hydrocarbon groups in the testing of liquid fuels Silica FIA fine (according to DIN and ASTM D T) mm Silica FIA coarse mm Silica E for the determination of the total amount of combustible organic substances in waste gases according to Ixfeld and Buck Silica E, mm mesh

37 Packings for CLC Packings for CLC Other inorganic adsorbents for low pressure column chromatography Aluminium oxide Aluminium oxide has been used for many years as an adsorbent in column chromatography. The raw materials for the production of chromatography grade aluminium oxides are different aluminium hydroxides, e.g. hydrargillite. The dehydration temperature is between 400 and 500 C. After dehydration the oxide has to be packed air-tight as quickly as possible to retain the activity grade I. Activity grades of aluminium oxide are between I and V. For an exact adjustment of activities II to V defined amounts of water are added to aluminium oxide of activity grade I. The resulting dry mixtures are left in a closed system until the added water is distributed evenly by surface diffusion and isothermal distillation. With occasional stirring this equilibrium is always reached after 24 hours. In tightly closed containers the activity grade is maintained for years. The following procedure by G. Hesse [Z. anal. Chem. 211 (1965) 5] is a useful rapid test for the activity. In a test tube a few ml of a 1% solution of triphenylchloromethane in absolute benzene are added to a sample to the aluminium oxide (about 1 g). If the sample turns yellow, it still has activity I. A water content as low as 0.5% will inhibit the colouration. Our aluminium oxides for low pressure column chromatography have activity grade I. On request, basic aluminium oxide is also available with other activity grades. Activity of oxidic adsorbents as a function of water content Activity grade Water content in % Aluminium oxide Silica I II 3 10 III 6 12 IV V Aluminium oxide 90 basic 1 kg 5 kg 25 kg ph 9.5 ± 0.3, activity grade I, particle size mm, specific surface (BET) ~ 130 m 2 /g Aluminium oxide 90 basic Aluminium oxide 90 neutral ph 7 ± 0.5, activity grade I, particle size mm, specific surface (BET) ~ 130 m 2 /g Aluminium oxide 90 neutral Aluminium oxide 90 acidic ph 4 ± 0.3, activity grade I, particle size mm, specific surface (BET) ~ 130 m 2 /g Aluminium oxide 90 acidic

38 Packings for CLC Packings for CLC Other inorganic adsorbents for low pressure column chromatography Kieselguhr Kieselguhr is a naturally occurring amorphous silicic acid of fossil origin, which is also known as diatomaceous earth or diatomite. Deposits are found in many parts of the world and were formed in recent geological times (quaternary and tertiary) from dead single-celled plants, the diatoms, the silica shells of which sank to the bottom of oceans or seas during very long periods of time forming more or less thick sediments. For application in chromatography, kieselguhr requires thorough purification which usually involves several steps and can be different for different origins of the raw material. During this clean-up procedure the porous structure must remain intact, although a decrease in surface area and a Kieselguhr adsorbents FLORISIL FLORISIL is a highly selective adsorbent which is often used in preparative and analytical chromatography. It is a white, very hard granular magnesia silica gel with the following composition: MgO 15.5 ± 0.5% SiO ± 0.5% Na 2 SO 4 max. 1.0% Typical application of FLORISIL adsorbents sample preparation (see chapter Solid phase extraction, page 213) clean-up of pesticide residues separation of chlorinated pesticides isolation of steroids, sex hormones etc. isolation of antibiotics separation of lipids widening of pores cannot be completely avoided. The surface area can decrease from m 2 /g of natural kieselguhr to 1 5 m 2 /g. Compared to silica, a small surface of low activity is available for the chromatographic separation. The application of kieselguhr in partition chromatography is a consequence of this structure. Due to its inactivity kieselguhr is often impregnated with various substances (paraffin, silicone oil, undecane) and used for reversed phase chromatography. We supply several grades of kieselguhr manufactured by Johns-Manville. They are narrowly classified with homogeneous particle size distributions and high purity. Designation rel. purification factor rel. flow rate 1 kg 5 kg Filter-Cel Standard Super -Cel Hyflo Super-Cel Celite Celite Celite FLOREX clay adsorbents This is a naturally occurring mineral which consists of magnesium aluminium silicate with a three-dimensional chain structure. Some grades are used as fillers or thickeners in the production of varnishes, others are applied as adsorbents. The RVM grades strongly adsorb polar molecules from solutions and are thus used for analytical purposes. Typical application of FLOREX RVM grades Adsorption of polar molecules from solutions hydrocarbon group analysis vitamin analysis FLOREX and FLORISIL are manufactured by Floridin Company, USA and registered trademarks of this company. Designation Application Particle size 1 kg 5 kg Florisil magnesium silicate, MgO 15.5 ± 0.5% SiO ± 0.5% Na 2 SO 4 max. 1.0% Florisil standard 60/100 mesh 0.15/0.25 mm Florex clay adsorbents magnesium aluminium silicate Florex S-RVM mm hydrocarbon group analysis according to mesh ASTM D T Florex S-RVM mm recommended for vitamin analysis mesh

39 Packings for CLC Packings for CLC Polyamide adsorbents for low pressure column chromatography It has long been known that strong hydrogen bonds can be formed between phenolic hydroxyl groups and amide bonds. These secondary valence forces allow the formation of defined complexes, e.g. between phenol and urea in the ratio 2:1 or between phenol and diketopiperazine. This effect which has been extensively described in the chemical literature should also enable the separation of phenolic substances on high-molecular polymers containing amide bonds. As commercial products polyamides such as Perlon (polycaprolactam, polyamide 6) and Nylon (polyhexamethylene diamine adipate, polyamide 66) are suited for this purpose. Due to the cross-linking via hydrogen bonds they are sufficiently insoluble in hydrophilic solvents like methanol, ethanol, acetone and dimethylformamide, but still capable of swelling. The distribution curve of phenol between polyamide 6 and water is linear until almost one third of the peptide bonds of the polyamide are saturated. This constant distribution coefficient over a wide range of concentrations is ideal for chromatographic separations. First attempts to use polyamide as packing for column chromatography were described by Carelli and co-workers and by Grassmann and co-workers. H O N H 2 C H 2 C H 2 C C H 2 C Polyamide 6 as stationary phase for the separation of phenols C CH 2 CH 2 N CH 2 CH 2 O H H O H 2 C N H 2 C H 2 C C H 2 C stationary phase C CH 2 CH 2 CH 2 N CH 2 O H H O mobile phase The affinity for polyamide increases in the series phenol, resorcinol, phloroglucinol, however, it decreases in the series phenol, pyrocatechol, pyrogallol. As a rule of thumb one can say that a second or third hydroxyl group in m or p position of an aromatic hydrocarbon increases the adhesion to polyamide, while in o position it decreases the bonding. In resorcinol and hydroquinone obviously both hydroxyl groups can simultaneously interact with two different amide bonds of the polyamide resulting in a stronger retention of the compounds compared to phenol. The hydroxyl groups of the pyrocatechol on the contrary have to compete for the same amide group. The second hydroxyl group strengthens the affinity for the usual aqueous eluent and thus additionally increases the migration speed. A partial saturation by intramolecular hydrogen bonds also may be important. Besides number and position of the hydroxyl groups of an aromatic compound the eluent has an important effect on the affinity of a phenol for polyamide. Depending on the ability of a solvent to compete with the hydrogen bonds between polyamide and phenol or to develop own interactions with the compound in question rapid desorption will occur. The desorption ability increases in the following sequence: water < methanol < acetone < diluted sodium hydroxide solution < formamide < dimethylformamide. The high elution strength of the dimethylformamide is probably due to the fact, that it possesses a CO N group itself and can thus form the same interactions with phenolic substances as the polyamide, i.e. it is the strongest hydrogen acceptor. The chromatography of phenolic compounds on polyamide has been frequently applied for the isolation and structure determination of different natural products. Carboxylic acids are also bonded to polyamide via hydrogen bonds. The affinity for monocarboxylic is still low. Dicarboxylic acids and aromatic acids are retained stronger, especially if the aromatic part of the molecule is larger. Strong affinity for polyamide is found with numerous aromatic nitro compounds. The interaction between the nitro compound and polyamide corresponds to a reaction of a Lewis acid with a base. This is why separations of aromatic nitro compounds on polyamide can be considered as ion exchange. For this reason elution in sharp bands requires an eluent with good buffering properties. This principle has been successfully applied for the separation of dinitrophenylamino acids which are formed in the determination of amino terminals of peptides and proteins on polyamide columns. Quinones are irreversibly bonded to polyamide due to the free amino groups of the adsorbent. Designation Particle size 1 kg 5 kg Polyamide 6 ε-aminopolycaprolactam Polyamide CC 6, < 0.07 mm < 0.07 mm Polyamide CC 6, mm mm Polyamide CC 6, mm mm

40 Packings for CLC Packings for CLC Cellulose adsorbents for low pressure column chromatography Unmodified cellulose Analytical data of native cellulose powders Parameter ff residue on ignition at 850 C < 1500 ppm < 200 ppm < 200 ppm iron [Fe] < 20 ppm < 2 ppm < 2 ppm copper [Cu] < 5 ppm < 1 ppm < 1 ppm phosphate [P] < 7 ppm < 2 ppm < 2 ppm methylene chloride < 0.20% < 0.15% < 0.02% extract average degree of polymerisation fibre length (85%) µm µm µm specific surface about 6500 about 5500 about 5500 acc. to Blaine cm 2 /g cm 2 /g cm 2 /g Cellulose powders for column chromatography are used for qualitative and preparative separations and purifications. Often it is necessary to wash the column packing with the desired eluent until the eluate is colourless and can be evaporated without residue. We supply cellulose powders of different grades and purities which can considerably shorten or even avoid the above-mentioned washing procedure depending on the separation required. For separations which require an extreme degree of purity of the column, however, washing with the eluent is recommended even for the prepurified cellulose. For economical reasons a commercial product cannot be washed with all organic eluents. Designation Particle size Cat. No. 50 g 1 kg 5 kg 25 kg Cellulose 100 / 2100 native fibrous cellulose, unmodified Cellulose Cellulose Cellulose 2100ff (cellulose 2100 defatted) Cellulose AVICEL microcrystalline cellulose, residue on ignition max. 0.01%, ph value of a 10% aqueous suspension Cellulose AVICEL ~ 38 µm Carboxymethyl cellulose 2100 CM R-O-CH 2 COOH cellulose ion exchanger, exchange capacity ~ 0.7 mval/g Cellulose 2100 CM Diethylaminoethyl cellulose 2100 DEAE R-O-C 2 H 4 -N(C 2 H 5 ) 2 cellulose ion exchanger, exchange capacity ~ 0.7 mval/g Cellulose 2100 DEAE Cellulose TRIACEL microcrystalline cellulose, acetylated, 40% acetyl content, recommended for enantiomer separation Cellulose TRIACEL Cellulose TRIACEL Microcrystalline cellulose Microcrystalline cellulose AVICEL for column chromatography is characterised by its very high purity. It is manufactured by hydrolytic degradation of refined pulp and cotton linters. The mean particle size is 38 µm with a residue on ignition below 0.01%. The ph value of a 10% aqueous suspension is

41 Packings for CLC Packings for CLC Cellulose adsorbents for low pressure column chromatography Cellulose ion exchangers Cellulose ion exchangers consist of cellulose with acidic or basic functions bonded via ether or ester bridges. Due to the wide network of the cellulose ion exchangers, which results from the fibre structure, most of the substituents are easily accessible. Even large hydrophilic molecules like e.g. proteins can diffuse through the swollen hydrophilic cellulose matrix. The distances between the active groups in cellulose exchangers are relatively large (about 50 Å). For this reason they are especially suited for larger molecules like e.g. proteins. Additionally, due to the larger distances of the active groups, binding occurs only at one or very few sites, allowing selective desorption under very mild conditions compared to resin exchangers. Last but not least, due to these characteristics cellulose ion exchangers are very valuable tools for the separation, purification and isolation of labile substances in biochemistry. Carboxymethyl (CM) cellulose R O CH 2 COOH CM cellulose prepared by reaction of alkali cellulose with monochloroacetic acid is a weak cation exchanger which has its highest exchange capacity in the ph range 4 5. As indicated in the formula above, CM cellulose is a monofunctional ion exchanger. It has an exchange capacity of about 0.7 mval/g and can absorb almost its own weight of proteins. Main fields of application: neutral and basic proteins, basic amino acids and hormones Diethylaminoethyl (DEAE) cellulose R O C 2 H 4 N(C 2 H 5 ) 2 DEAE cellulose prepared by reaction of alkali cellulose with 2-chloro-1-diethylamino-ethane hydrochloride is a weakly basic anion exchanger. The basic group is monofunctional. Our DEAE cellulose has an exchange capacity of about 0.7 mval/g. DEAE cellulose is the most often used cellulose ion exchanger. Main fields of application: separation, purification and isolation of proteins, enzymes and hormones. Cellulose triacetate as enantioselective packing Cellulose triacetate is one of the oldest stationary phases in liquid chromatography. G. Hesse and R. Hagel were the first to use this phase for chiral separations [Chromatographia 6 (1973) 277]. They could separate the racemate of Troeger's base by columns liquid chromatography as well as by thin layer chromatography. However, an important prerequisite for successful enantiomer separations is the use of a completely triacetylated cellulose. Very good results are obtained with microcrystalline cellulose, if it is acetylated in situ without going into solution. A cellulose triacetate prepared like this is not only suited for separation of racemates, but also can be used for many other chromatographic separations, showing unusual specificities, which are of theoretical and in some cases of preparative interest [G. Hesse and R. Hagel, Chromatographia 9 (1979) 62]. The helical structure of the stationary phase allows enantiomer separations of compounds based on steric, i.e. inclusion effects even if the solute molecules do not possess aromatic ring systems for π - π interactions or functional groups for hydrogen bonding (e.g. plain aliphatic hydrocarbons). Numerous pharmaceuticals can be separated, among them barbiturates, ketamines, aziridines, sulphoxides, mandelic acid derivatives, pyrrolidine derivatives, azepines, imidazole derivatives, isoquinolines and hydantoins. Basic substances are chromatographed as free bases, not as salts. It is very important to remember that microcrystalline cellulose swells to a different degree in organic solvent mixtures, e.g. by about 40% of its volume in ethanol/water (95:5). In practice this means that cellulose triacetate columns should only be operated with the eluent in which the adsorbent was swollen and packed. Also, during elution the pressure should not exceed a fraction of the packing pressure. Our cellulose triacetate is a completely acetylated microcrystalline cellulose, which is available in two particle size fractions. 162

42 Packings for CLC Packings for CLC Flash chromatography Separations fast as lightning with the MACHEREY-NAGEL flash chromatography system fast and economic methods for the synthesis laboratory ideal for the separation of compounds up to gram quantities no expensive equipment required in an ideal way transfers results from TLC to CLC Flash chromatography is a fast and economic column liquid chromatographic separation method. The primary field of application is the prep-scale purification of organic compounds, as originally developed by W. C. Still et al. in 1978 [W. C. Still, M. Kahn and A. Mitra, J. Org. Chem. 43 (1978) 2923]. The principle is that the eluent is, under gas pressure (normally nitrogen or compressed air) rapidly pushed through a short glass column with large inner diameter. The glass column is packed with an adsorbent of defined particle size. The most used stationary phase is silica gel µm, but obviously packings with other particle sizes can be used as well. Particles smaller than 25 µm should only be used with very low viscosity mobile phases, because otherwise the flow rate would be very low. Normally gel beds are about 15 cm high with working pressures of bar. Originally only unmodified silica was used as the stationary phase, so that only normal phase chromatography was possible. In the meantime, however, and parallel to HPLC, reversed phase materials are used more frequently in flash chromatography. The flash chromatography system consists of: glass columns from 20 mm ID x 200 mm length to 40 mm ID x 450 mm length, all complete with adaptor and teflon tap. The columns are fitted with a polyethylene net to protect against bursting eluent reservoirs, 1 l or 2 l content, with adaptor, covered with a protective plastic sleeve for burst protection; the protective sleeve also prevents build-up of UV-induced radicals in the eluent pressure gauge for controlling flow rates Furthermore, two different flash chromatography kits are available. Each kit contains a glass column, an eluent reservoir, silanised glass fibre wadding and some sea sand. In addition to detailed instructions for use, 100 g MACHEREY- NAGEL silica 60 with µm particle size are also included. 163

43 Packings for CLC Packings for CLC Flash chromatography MACHEREY-NAGEL flash adsorbents highest quality for completely reproducible separations The basic prerequisite for successful separations is choice of the proper adsorbent. The most important stationary phase in column chromatography is silica. Strict quality control during production and the following precise fractionation guarantee the high quality and batch to batch reproducibility of our silicas. In addition to our well known standard grade silica, we also offer POLYGOPREP, which is a very pure silica gel with a specially low content of metal ions. POLYGOPREP is available as unmodified silica, and also as RP phases and with chemically bonded polar phases, thus considerably broadening the range of applications for flash chromatography. Designation Pack of Cat. No. Flash chromatography kits Flash chromatography kit I, consists of 1 glass column 20 mm ID x 400 mm, one 1 l eluent reservoir, 100 g silica 60 (40 63 µm), sea sand, silanised glass fibre wadding Flash chromatography kit II, consists of 1 glass column 40 mm ID x 450 mm, one 2 l eluent reservoir, 100 g silica 60 (40 63 µm), sea sand, silanised glass fibre wadding 1 kit kit Flash chromatography columns, complete with adaptor and teflon tap 20 mm ID x 200 mm length 1 column mm ID x 400 mm length 1 column mm ID x 200 mm length 1 column mm ID x 400 mm length 1 column mm ID x 300 mm length 1 column mm ID x 400 mm length 1 column mm ID x 300 mm length 1 column mm ID x 450 mm length 1 column Accessories for flash chromatography columns Eluent reservoir 1 l with adaptor Eluent reservoir 2 l with adaptor Pressure gauge Sea sand, acid washed and calcined 1000 g Glass fibre wadding, silanised 25 g

44 Accessories for stainless steel HPLC columns Accessories for HPLC Accessories for HPLC Stainless steel columns are most frequently used in HPLC. The material is corrosion resistant, pressure stable and easy to work mechanically. Stainless steel empty columns for HPLC are available on request. Designation Pack of Cat. No. Accessories for analytical stainless steel columns Accessories and replacement parts for EC columns with 2, 3, 4 and 4.6 mm ID 1/16 nut for connecting 1/16 capillaries /16 ferrule /16 end cap, plastic EC fitting adaptor EC column head (nut) EC PTFE sealing ring part sealing combination for EC columns 5 kits NUCLEOGEL column accessories Frits 2 µm for 4.6 mm ID columns Frits 2 µm for 7.7 mm ID columns Column connection nuts for 1/16 capillaries Ferrules for 1/16 capillaries Union for columns Column end plugs Accessories for preparative stainless steel columns Accessories for Standard-Prep columns with 10 mm ID SP end fitting for 10 mm ID, complete Wire screen, stainless steel, for 10 mm ID, /2 OD Glass fibre filter 85/90 BF, 1/2 OD Accessories for Standard-Prep columns with 21 mm ID SP nut 21 mm Accessories for VarioPrep columns with 10 mm ID VP plunger fitting 10 mm VP nut 10 mm VP sealing element set 10 mm 1 set VP sealing ring set 10 mm 1 set VP Inert sealing combination 10 mm 1 set Accessories for VarioPrep columns with 21 mm ID VP plunger fitting 21 mm VP nut 21 mm VP sealing element set 21 mm 1 set VP sealing ring set 21 mm 1 set VP Inert sealing combination 21 mm 1 set

45 Accessories for HPLC Accessories for HPLC Accessories for stainless steel HPLC columns Stainless steel capillary tubing and capillary accessories for LC We supply 1 / 16 capillaries as coils (1, 3 or 33 m) or as tubes cut to exact length with smooth ends (ready-to-use). Description Pack of Cat. No. Description Pack of Cat. No. Capillary tubing in coils Capillary accessories 33 m x 1/16 x 0.25 mm 1 coil /16 capillary tubing cutter m x 1/16 x 0.5 mm 1 coil (knife file) 3 m x 1/16 x 0.25 mm 1 coil Spare knife file m x 1/16 x 0.5 mm 1 coil Cutter for 1/16 capillaries m x 1/16 x 0.12 mm 1 coil m x 1/16 x 0.25 mm 1 coil m x 1/16 x 0.5 mm 1 coil Stainless steel capillary tubing, cut pieces ready-to-use Capillary unions 100 mm x 1/16 x 0.25 mm 50 mm x 1/16 x 0.12 mm 2 tubes Type 1 1 union mm x 1/16 x 0.12 mm 2 tubes mm x 1/16 x 0.12 mm 2 tubes mm x 1/16 x 0.12 mm 2 tubes mm x 1/16 x 0.25 mm 5 tubes Type 2 1 union mm x 1/16 x 0.25 mm 5 tubes mm x 1/16 x 0.25 mm 5 tubes mm x 1/16 x 0.5 mm 5 tubes mm x 1/16 x 0.5 mm 5 tubes Type 3 1 union mm x 1/16 x 0.5 mm 5 tubes mm x 1/32 x 0.12 mm 2 tubes mm x 1/32 x 0.12 mm 2 tubes mm x 1/32 x 0.12 mm 2 tubes mm x 1/32 x 0.25 mm 2 tubes mm x 1/32 x 0.25 mm 2 tubes Eluent filters, stainless steel 200 mm x 1/32 x 0.25 mm 2 tubes for 1/16 tubing 2 µm frit mm x 1/32 x 0.5 mm 2 tubes for 1/16 tubing 10 µm frit mm x 1/32 x 0.5 mm 2 tubes for 1/8 tubing 2 µm frit mm x 1/32 x 0.5 mm 2 tubes for 1/8 tubing 10 µm frit

46 Accessories for HPLC Accessories for HPLC PEEK column accessories PEEK (= polyether ether ketone) is a high performance polymer belonging to the group of polyarylether ketones (PAEK), which meets all requirements of HPLC columns with respect to chemical resistance and mechanical stability. In some fields of application in HPLC, like e.g. in ion chromatography and chromatography of biopolymers, PEEK fulfils the requirements for a nonmetallic material. All fittings can be tightened by hand. O O The following table summarizes the available PEEK products. O C n PEEK Description Pack of Cat. No. PEEK accessories 1/16 PEEK fingertight fitting, part combination nut + ferrule 1/16 PEEK fingertight nut /16 PEEK ferrule for Cat. No /16 PEEK hex nut /16 stainless steel handtight nut /16 PEEK double ferrule for Cat. Nos and /16 PEEK union, both sides inner threads, equipped with 2 fingertight nuts and double ferrules /16 PEEK union, both sides inner threads, however without nuts and without ferrules 1/16 union with outer threads, consists of 1/16 capillary tubing and 2 fingertight fittings PEEK capillaries PEEK standard capillaries OD ID [mm] Length 1/ m / m / m / m / m /8 1,6 1 m

47 Accessories for HPLC Accessories for HPLC PEEK column accessories Description Pack of Cat. No. PEEK Telephone capillaries, complete with fingertight fittings 1/ cm / cm / cm / cm / cm / cm / cm / cm / cm Tools for PEEK capillaries Guillotine cutter for PEEK and PTFE capillaries Clean-Cut cutter for different capillary outer diameters Injectors and switching valves for HPLC Valco Cheminert HPLC injection valves are operated in two positions: load and inject. Valves with 0.25 mm orifice are recommended for microbore applications, while for analytical routine analysis valves with 0.4 mm bore are used. Valve type C1 The classical HPLC injection valve with through-the-handle injection allows choice between the partial filling method, in which the injection volume is determined by a syringe, and full-loop injection, in which the volume is determined by the size of the sample loop. The Cheminert design prevents any contact between the needle and the stator or rotor faces, eliminating the possibility of scratching these critical sealing surfaces. Because the handle is integral to the design, all C1 valves are manual. Nuts and ferrules, position feedback adaptor and 20 µl sample loop (5 µl for microbore valves) are included. Valves with PAEK stator are supplied with PEEK fittings and sample loops. Stator material Analytical (0.4 mm bore) Microbore (0.25 mm bore) Valco code Cat. No. Valco code Cat. No. C1 valves with 6 ports 1/16" fittings, P max : 5000 psi, T max : 75 C (PAEK stator 50 C) Nitronic 60 C C12006 C C11006 PAEK C C12046 C C11046 Titanium C C12036 C C

48 Accessories for HPLC Accessories for HPLC Injectors and switching valves for HPLC Valve type C2 This valve can be used as injector or as a switching valve. It is available as 4, 6, 8 or 10 port version. Injection is made directly into the valve head using a fill port (Cat. No. 724CLFP). The model C2 is available with all VICI actuation options (manual, pneumatic, electric). With 6 port valves, a loop of material similar to the stator is included (5 µl for microbore, 20 µl for analytical valves). The manual version is supplied with position feedback adaptor. We can supply any valve or fitting from the complete Valco programme. For detailed information about e.g. multiposition valves, valve actuators and controls as well as in-line filters, fittings made from metal or different polymer materials please ask for the Valco Cheminert catalogue. Analytical (0.4 mm bore) Microbore (0.25 mm bore) Actuation Valco code Cat. No. Valco code Cat. No. C2 valves with 4 ports 1/16" fittings, P max : 5000 psi (liq), T max : 75 C, stator material Nitronic 60 Manual C C22004 C C21004 Pneumatic C2-2004A 724C22004A C2-1004A 724C21004A Electric (standard) C2-2004E 724C22004E C2-1004E 724C21004E Electric (micro) C2-2004EH 724C22004EH C2-1004EH 724C21004EH C2 valves with 6 ports 1/16" fittings, P max : 5000 psi (liq), T max : 75 C, stator material Nitronic 60 Manual C C22006 C C21006 Pneumatic C2-2006A 724C22006A C2-1006A 724C21006A Electric (standard) C2-2006E 724C22006E C2-1006E 724C21006E Electric (micro) C2-2006EH 724C22006EH C2-1006EH 724C21006EH C2 valves with 8 ports 1/16" fittings, P max : 5000 psi (liq), T max : 75 C, stator material Nitronic 60 Manual C C22008 C C21008 Pneumatic C2-2008A 724C22008A C2-1008A 724C21008A Electric (standard) C2-2008E 724C22008E C2-1008E 724C21008E Electric (micro) C2-2008EP 724C22008EP C2-1008EP 724C21008EP C2 valves with 10 ports 1/16" fittings, P max : 5000 psi (liq), T max : 75 C, stator material Nitronic 60 Manual C C22000 C C21000 Pneumatic C2-2000A 724C22000A C2-1000A 724C21000A Electric (standard) C2-2000E 724C22000E C2-1000E 724C21000E Electric (micro) C2-2000EP 724C22000EP C2-1000EP 724C21000EP C2 valves are also available with PAEK or titanium stator. 169

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