HPLC Columns-ProntoSIL Contents ProntoSIL C18-EPS column 197 ProntoSIL C30-EPS column 201 ProntoSIL 300Å Wide Pore column 203 ProntoSIL Chiral AX colu

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1 FUTECS your chromatography specialist HPLC Columns : Pronto SIL C18-EPS Column C30-EPS Column 300Å Wide Pore Column Chiral AX Column 195

2 HPLC Columns-ProntoSIL Contents ProntoSIL C18-EPS column 197 ProntoSIL C30-EPS column 201 ProntoSIL 300Å Wide Pore column 203 ProntoSIL Chiral AX column 204 ProntoSIL column 종류와특성 232 ProntoSIL C18(C8) ace-eps Columns(60A, 120A, 200A, 300A) ProntoSIL C18 H Columns(60A, 120A, 200A, 300A) ProntoSIL C18(C8) SH Columns(60A, 120A, 200A, 300A) ProntoSIL C18 AQ Columns(60A, 120A, 200A, 300A) ProntoSIL Enviro C18 Columns(5um, 6um, 7um) ProntoSIL Hypersorb ODS Columns(3um, 5um) ProntoSIL Spheribond ODS Columns(3um, 5um, 10um) ProntoSIL Eurobond C18 Columns(5um) ProntoSIL C30 Columns(3um, 5um, 10um) ProntoSIL AMINO Columns(3um, 5um, 10um) ProntoSIL Silica Columns(3um, 5um) ProntoSIL Phenyl Columns(3um, 5um, 10um) ProntoSIL CN Columns(3um, 5um, 10um) ProntoSIL DIOL Columns(3um, 5um, 10um) Applications Applications Applications Applications Applications Applications Applications 참고자료 250 HPLC column 고정상의종류 Column 의물리적성질 196

3 FUTECS your chromatography specialist ProntoSIL C18-EPS Reversed-Phase HPLC Columns Using New Polar Embedded Phases to Optimize Reversed Phase Separations Figure 1 High Polarity Stationary Phases Offer Alternate Selectivity to Base Deactivated Phases Column A: Base deactivated C18 column with low polarity 1 Column B: C18 column with high polarity due to silanol activity 2 α = Time (min) Introduction Base deactivated stationary phases have been a real asset to chromatographers who develop HPLC separations of polar compounds. Columns packed with these base-deactivated phases provide better peak shape, increased column efficiency, and improved lot-to-lot reproducibility, especially when separating bases. However, there are occasions when a typical base deactivated phase may not provide an optimum separation. Manufacturers of base deactivated columns try to minimize interactions between polar solutes and silanol groups on the surface of the stationary phase support. This produces stationary phases with low polarity and these low polarity phases will all have similar selectivity for polar compounds. This means that if one brand of base deactivated column lacks selectivity to adequately separate a pair of polar solutes, other brands of base deactivated columns will also probably lack adequate selectivity. To optimize the separation, a more polar stationary phase may be necessary. Unfortunately, stationary phases that have polar characteristics due to silanol activity will often exhibit poor peak shape and uncertain reproducibility when separating polar compounds (Figure 1). To solve this problem, column manufacturers have developed a new type of base deactivated stationary phase with polar groups, such as amides or carbamates, embedded in the bonded phase (Figure 2). These polar embedded phases provide polar selectivity without the poor chromatographic performance associated with stationary phases that have high silanol activity. 1 α = 3.48 Figure 2 Polar Embedded Phases Time (min) Mobile Phase : 55% CH 3 OH 45% H 2 O 2 alkyl bonded phase Polar Group Examples of Polar Embedded Phases: Chromegabond ProTech-RP Discovery RP-Amide C16 Hypersil HyPURITY Advance Keystone Prism ProntoSIL C18-EPS Symmetry Shield Zorbax Bonus-RP : Analytes 1. Acetylsalicylic acid 2. Salicylic acid Column B (significant silanol activity) provides greater selectivity than Column A for these polar solutes but at a cost of poorer peak shape for salicylic acid. Silica By embedding a polar group, such as an amide or carbamate, in the bonded phase a new type of base deactivated column with polar selectivity can be created

4 HPLC Columns Strategy for Developing Reversed Phase Separations of Ionic Compounds Table 1 provides a brief outline of a typical strategy used to develop an isocratic separation of polar compounds. As mentioned before, typical base deactivated columns sometimes fail to provide adequate selectivity for mixtures of polar compounds. This is where polar embedded phases should be used. The polar characteristics of these special base deactivated columns will often provide significantly better selectivity for mixtures of acids and bases. Therefore, if during the process of developing a separation you find that a typical C18 or C8 column does not provide adequate selectivity, your next step should be to select a polar embedded phase column to evaluate. Figure 3 Solvent Elution Strength Comparison Table 1 Strategy for Developing Reversed Phase Separations of Polar Compounds Select a base deactivated C18 or C8 column. Use a mobile phase consisting of acetonitrile and M potassium phosphate at a ph less than 3.0. Adjust the amount of acetonitrile in the mobile phase until the k values of all peaks are between 1 and 10. Evaluate the selectivity, and if it is unacceptable, (i.e., not all peaks of interest are adequately separated), substitute methanol for acetonitrile and again evaluate selectivity. Figure 4 will help you calculate the amount of methanol needed in the mobile phase to keep the elution strength constant. If selectivity is still unacceptable, choose a different stationary phase to evaluate. This can be a different brand of C18, but your best bet is to try a different bonded phase chemistry such as CN, Phenyl, or a polar embedded phase. Once the selectivity is acceptable, optimize the separation by adjusting the mobile phase strength (% organic solvent composition) and flow rate until all peaks are adequately separated (Rs > 1.5) and the system back pressure and overall separation time is suitable for the application. In a rigorous method development process, mobile phase ph, mobile phase additives (buffer concentration, ion pair reagents, amine modifiers, etc.), column temperature, and column configuration should also be evaluated and optimized. % Acetonitrile % Methanol Figure 4 Polar Embedded Phases Offer Alternate Selectivity to Typical Base Deactivated Phases Typical base deactivated column (ProntoSIL-C18H) Mobile Phase: : 6 65% Acetonitrile 35% M Phosphate buffer, ph Uracil 2. Pyridine 3. Phenol 4. N,N-Dimethylaniline 5. p-butylbenzoic acid 6. Toluene Time (min) 2 Base Acid Base Polar embedded phase column (ProntoSIL C18-EPS) Acid Acetonitrile is a stronger solvent than methanol for reversed phase HPLC. This nomograph will help you keep the mobile phase elution strength constant when you switch from acetonitrile to methanol. For example, you will need about 62% methanol in the mobile phase to equal the eluting strength of 50% acetonitrile Time (min) In general, polar embedded phases will retain acids longer and bases slightly less than typical base deactivated columns. 198 Bischoff

5 FUTECS your chromatography specialist Using The Polar Selectivity of Polar Embedded Phases to Optimize a Separation Figure 6 Subtle Differences in Stationary Phase Selectivity Can Be Useful in Optimizing HPLC Separations 1 4,5 Typical C18 base deactivated column Columns packed with polar embedded phases are used in the same way as typical reversed phase columns and the separation strategy outlined in Table 1 is also appropriate for these columns. However, you should expect significant differences in selectivity from polar embedded phases due to their polar selectivity. The polar selectivity of polar embedded phases comes from the interaction between the amide or carbamate group and polar solutes. In general you can expect that acidic compounds will be retained longer and basic compounds will be retained slightly less on polar embedded phases compared to typical reversed phase columns (Figure 4). Sometimes the selectivity differences can be dramatic, as shown in Figure 5, and sometimes they may be more subtle but still significant in optimizing a separations, as shown in Figure Time (min) Polar embedded phase with enhanced polar selectivity (ProntoSIL C18 EPS) Mobile Phase: : Time (min) % Acetonitrile 50% Water DNPH s of Aldehydes and Ketones 1. DNPH 2. Formaldehyde 3. Acetaldehyde 4. Acetone 5. Acroleine 6. Propanale 7. Butanale The polar selectivity offered by polar embedded phases will often provide a better separation. In this separation of DNPH s of aldehydes and ketones, compounds 4 and 5 co-elute on a typical base deactivated phase, but are well separated on a polar embedded phase. 7 7 FIGURE 5 Polar Embedded Phases Can Offer Dramatic Improvements in Selectivity Hypersil BDS C18 α = ProntoSIL C18 EPS α = Mobile Phase: : 1 The ProntoSIL C18-EPS with enhanced polar selectivity provides significantly better selectivity for this pair of polar solutes than a typical base deactivated column (Hypersil BDS C18). 2 65% CH 3 CN 35% Phosphate buffer, ph N,N-Dimethylaniline 2. p-butylbenzoic acid Time (min) Time (min) 2 Improved Peak Shape Most polar embedded phases use high purity silica as the stationary phase support and thoroughly cover the silica with bonded phase to reduce interaction between acidic silanols and polar solutes. This in itself would produce a stationary phase with good peak shape for basic compounds, but polar embedded phases also have the advantage of further deactivating the stationary phase support by means of the amide or carbamate groups. Although the mechanism is not fully understood, the prevailing view is that the polar embedded groups interact with unbonded silanols on the silica stationary phase support and thereby block them from interacting with polar solutes. The effect is similar to adding an amine modifier to the mobile phase. This polar shielding, as some manufacturers call it, gives these phases excellent peak shape for basic compounds over a broad ph range (Figure 7). Figure 7 Polar Embedded Phases Provide Excellent Peak Shape for Basic Compounds Mobile Phase: Analyte: 80% Methanol, 20% M Phosphate buffer, ph 6.0 Amitriptyline Hypersil BDS C18 ProntoSIL C18 EPS The amide group of the ProntoSIL C18-EPS shields the silica surface and prevents solutes from interacting with silanol groups. The result is exceptionally good peak shape for even difficult basic compounds

6 HPLC Columns Figure 8 Phase Collapse Figure 10 Bonded Phase on the ProntoSIL C18-EPS is Secured Through Dual Siloxane Bonds Fully Extended Alkyl Phase Silica Surface H 2 O Organic Solvent Phase Collapse ProntoSIL C18 EPS with Dual Siloxane Bonds Typical Bonded Phase Silica Support Silica Support Si O Si Si O CH3 CH 3 Si OS i Polar C18 Phase Dual bonds resist acid hydrolysis s CH 3 C18 Phase Acid hydrolysis cleaves siloxane bond Si O Si Polar C18 Phase Si O CH3 Bonded phase unaffected CH 3 s Si OS i C18 Phase CH 3 Bonded phase lost Acid hydrolysis of the siloxane bond and the resulting loss of bonded phase (i.e., C18 Phase) is one of the major reasons for column failure. ProntoSIL C18-EPS uses unique bonding chemistry to secure the polar C18 phase to the silica support through dual siloxane bonds. The dual bonding inhibits the loss of bonded phase and makes these columns unusually stable. Silica Surface When operating with less than 10% organic modifier in the mobile phase, typical C18 and C8 phases are susceptible to phase collapse. Polar embedded phases do not have this problem and are preferred over typical base deactivated phases for high aqueous mobile phase conditions. Figure 9 Comparison of Retention of Some Popular Polar Embedded Phases 1 2 ProntoSIL C18-EPS 1 Symmetry Shield C Discovery Amide 7 7 Zorbax Bonus RP Polar Embedded Phases Are Preferred for High Aqueous Mobile Phase Conditions The alkyl bonded phase of typical C18 and C8 columns undergo what many researchers call phase collapse, or matting, when operating with less than 10% organic modifier in the mobile phase (Figure 8). As phase collapse progresses, the availability of the alkyl phase to interact with solutes decreases and retention time decreases. Polar embedded phases can be used with high aqueous mobile phases without the problem of phase collapse. The polar embedded groups permit the stationary phase surface to remain wet even under 100% aqueous mobile phase conditions. This keeps the bonded phase fully extended into the mobile phase, eliminates phase collapse, and facilitates the retention of highly water soluble compounds that may be poorly retained on typical reversed phase columns. Ion pair reagents can be avoided so that chromatographic conditions are simpler and methods are more rugged. Retention Comparison In general, polar embedded phase columns are less hydrophobic and therefore less retentive than typical reversed phase columns. ProntoSIL C18-EPS, however, is an exception. It provides similar retention as other C18 phases for neutral and hydrophobic compounds and slightly more retention for some highly water soluble compounds. Figure 9 provides a comparison of retention for four popular polar embedded phases Column: Mobile Phase: Flow Rate: : 4.6 x 150 mm 50% CH 3 CN 50% H2O 1.0 ml/min 1. Formaldehyde 5. Propanale 2. Acetaldehyde 6. Crotonaldehyde 3. Acetone 7. Butylraldehyde 4. Acroleine Most polar embedded phase columns are less hydrophobic, and thus less retentive than typical reversed phase columns. ProntoSIL C18-EPS, however, is an exception and provides comparable retention to other C18 phases. In this example, we see that ProntoSIL C18-EPS is the most retentive of these four polar embedded phases. Bonded Phase Stability There is no reason to believe that polar embedded phases are any less stable than typical C18 and C8 phases. Most manufacturers report stability data to support this. One column, Zorbax Bonus RP, uses unique silanes with bulky side groups to add stability to its bonded phase. Another column, ProntoSIL C18-EPS, uses dual siloxane bonds to increase bonded phase stability and actually demonstrates much greater durability than other C18 phases as well as other polar embedded phases (Figure 10). The next time you encounter poor selectivity while trying to separate polar solutes on a base deactivated phase, try one of these new polar embedded phases. You will be more likely to achieve a satisfactory separation with the polar embedded phase than with another brand of base deactivated column. 200 Bischoff

7 FUTECS your chromatography specialist ProntoSIL C30-EPS Reversed-Phase HPLC Columns Unique planar selectivity for carotenoids and structural isomers Excellent reproducibility Multi-valent silane bonding for enhanced stability Reversed Phase HPLC Columns for the Separation of Hydrophobic Structural Isomers ProntoSIL C30 HPLC columns are particularly recommended for the separation of hydrophobic, long-chain, structural isomers. They are often a better alternative to normal phase columns for the separation of isomers since they are not as sensitive as normal phase columns to water content of the mobile phase and are not as susceptible to column fouling. ProntoSIL C30 columns show significantly greater shape selectivity compared to C18 phases due to their rigid, highly ordered C30 alkyl groups (Figure 6). This shape selectivity advantage makes them the ideal HPLC column to use for the separation of carotenoids (Figure 7) and tocopherol derivatives (Figure 8). For best performance, C30 columns should be used at ambient temperature or lower. At elevated temperatures, the C30 alkyl chains will become less ordered and lose their shape selectivity (Figure 9). In addition, C30 phases should be used with mobile phases containing at least 20% organic modifier to avoid phase collapse. Although you can expect lower plate count on C30 phases compared to C18 phases, the ability of the C30 phases to separate isomers that are not possible to separate on C18 phases make them an attractive choice for many applications. C30 phases have been successfully used for the analysis of plant extracts, food, and biological tissues that contain structurally different carotenoids and mixtures of geometric isomers. They are also commonly used for separating stereoisomers of vitamin E and vitamin A, and for the separation of PAHs. Figure 6 Comparison of C30 and C18 Phases for the Separation of Lutein Stereoisomers Figure 7 HPLC Separation of Carotenoids Figure 8 HPLC Separation of Tocopherol Isomers C18 Mobile Phase: 76% Acetone 24% H 2 O 1, Time (min) 4,5 all-trans 9-cis Column: Mobile Phase: Flow Rate: Temperature: : ProntoSIL C30,4.6 x 250 mm 96% Methanol,4% Water 0.9 ml/min 25 C δ-tocopherol γ-tocopherol 2 β-tocopherol α-tocopherol 1 3 C Mobile Phase: 85% Acetone 15% H 2 O Time (min) Time (min) Flow Rate: Detector: : 1 ml/min UV at 450 nm 1) 13-Z lutein, 2) 13 -Z lutein, 3) all-e-lutein 4) 9-Z lutein, 5) 9 -Z lutein Column: Mobile Phase: Flow Rate: Temperature: ProntoSIL C30,4.6 x 250 mm 80% Methanol,20% TBME 1.4 ml/min 20 C By courtesy of Prof.Dr.K.Albert et al, University Tuebingen,Germany

8 HPLC Columns Figure 10 HPLC Separation of Steroids (Metabolites of Norethisterone Acetate) Figure 9 The Effect of Temperature on Shape Selectivity of C30 Phases Mobile Phase: Flow Rate: Detection: : 1 50% to 100% B,60 minutes A:Water B:Methanol 0.6 ml/min UV at 260 nm 2. p-butylbenzoic acid 1) 6-Keto-Norethisterone acetate 2) Estradiol 3) Norethisterone acetate By courtesy of Prof.Dr.K.Albert et al, University Tuebingen,Germany. 2 Time (min) 40 C 30 C 20 C 10 C 13-cis all-trans 9-cis Specifications Stationary Phase Support: Spherical,ultra-pure, Type B silica 3 and 5 micron particle size 200 Angstrom pore size 200 m2/gram surface area Bonded Phase: Polymeric bonding chemistry, non-endcapped C30 (Triacontylsilane) 18.5% carbon load Time (min) ProntoSIL C30 Ordering Information Dimensions Particle (mm) Size (μm) Part Number 2.0 x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS x H300PS050 Guard Cartridges (Guard Cartridge Holder required) 4.0 x 10 (5 pk) H300PS x 10 (5 pk) H300PS050 Guard Cartridge Holder Bischoff

9 ProntoSIL 300Å Wide Pore FUTECS your chromatography specialist ProntoSIL 300Å Wide Pore These wide pore phases are recommended for the separation of proteins and peptides. As with all ProntoSIL phases, ultra pure silica is used for the stationary phase support, and the latest bonding technology is used to provide rugged, reliable separations. C18, C8, and C4 bonded phase are available. Figure 14 Separation of Peptides on ProntoSIL C18 300H Figure 15 Separation of Tryptic Digest of Casein ProntoSIL C18 300H Column: Mobile Phase: Flow Rate: Detector: 4.6 x 75 mm, 3.5 μm Gradient: 0-40% B, 24 min A: Water with 0.1% TFA B: 70% Acetonitrile 30% Water with 0.1% TFA 1.0 ml/min UV, 220nm Temperature: 30 C : Tryptic digest of casein ProntoSIL C18 300H Time (min) Column: 4.6 x 250 mm, 5 μm Mobile Phase: Gradient: % B, 30 min A: Water with 0.1% TFA Time (min) B: 70% Acetonitrile 30% Water with 0.1% TFA Flow Rate: 1.5 ml/min Detector: UV, 220nm Temperature: 30 C : 1.Oxytocin 4. Eledoisi 2. Bradykinin 5. Neurotensin 3. Angiotensin II 6. Angiotensin I The wide pore ProntoSIL C18 300H column is recommended for the separation of peptides High efficiency ProntoSIL C18 300H 3 micron material packed in a short column provides high speed, high resolution separation of tryptic digests. Specifications for ProntoSIL 300Å Phases Phase End capped Particle Pore Surface Carbon Load Size (μm) Size Å Area (m 2 /g) (%) C H yes 3, C8 300 SH yes 3, C4 300 yes 3, ProntoSIL 300Å Wide Pore Ordering Information Dimensions Particle (mm) Size (μm) C H C8 300 SH C x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS x K185PS K080PS K040PS050 Guard Columns* 2.0 x K185PS K080PS K040PS x K185PS K080PS K040PS050 Guard Cartridge Holder Set *Requires Hardware Kit 203

10 HPLC Columns ProntoSIL Chiral AX HPLC Columns Multi-mode separation: Reversed phase, non-aqueous reversed phase (NARP), normal phase Two complementary phases: Separate enantiomers in the order R, S or S, R Stability: ph stable from 2-8 High loadability: Up to 50 mg sample per gram of stationary phase ProntoSIL Chiral AX Multi-modal, Complementary Chiral HPLC Columns The new ProntoSIL Chiral AX HPLC columns from Bischoff Chromatography can be described in one word; versatile. With Chiral AX, you now have the versatility to use any mobile phase, aqueous or organic, reversed phase or normal phase. Furthermore, Chiral AX now offers ph stability for chiral separations similar to that obtained with standard reversed phase columns. Since the objective of many chiral separations is isolation and purification of one enantiomer, Chiral AX offers both high loadability and the option of reversing elution order by using one of two complementary bonded phases. Description ProntoSIL Chiral AX HPLC columns are weak anion exchangers and are very well suited for the separation of acidic chiral analytes such as N-derivatized amino acids, amino sulfonic acids, amino phosphonic acids, acidic pharmaceuticals, arylcarbonic acid herbicides and N-derivatized peptides. Two chiral selectors are offered: carbamylated quinine (ProntoSIL Chiral AX QN-1) and carbamylated quinidine (ProntoSIL Chiral AX QD-1). The structure of these phases is show in Figure 11. Choose a Mobile Phase That s Right for Your ProntoSIL Chiral AX columns are multimodal in that they can be used in either reversed-phase, non-aqueous reversed phase or normal phase modes. Many chiral columns restrict the amount of organic or aqueous solvent allowed in the mobile phase. Because of the non-covalent bonding used in these other columns, use of solvents outside the recommended range can cause irreversible column damage. The chiral selective group used in Chiral AX is covalently bonded to the silica support and can be used in the same manner as a typical reversed phase column. These columns are well suited for use in high aqueous mobile phases making them the ideal choice for the separation of biological enantiomers such as amino acids and peptides. Figure 11 Structure of the Chiral AX Phases Figure 12 Separation of the Enantiomers of Allylglycine on Both Supports H N O O H 9 8 H N 1 S 3 R 4 S O S CH 3 silica ProntoSIL Chiral AX QN-1 ProntoSIL Chiral AX QD-1 N ProntoSIL Chiral AX QN-1: Carbamylated Quinine (8S, 9R) ProntoSIL Chiral AX QD-1: Carbamylated Quinidine (8R, 9S) Column: Mobile Phase: Flow Rate: 4.0 x 150 mm 99% Methanol 1% Acetic Acid 1 ml/min Temperature: 25 C Detection: UV 230 nm 204 Bischoff

11 FUTECS your chromatography specialist Achieve Complementary Separations The chiral selectivity of ProntoSIL AX is due to several possible mechanisms including steric interaction, hydrogen bonding, π-π interactions and ion pairing. These different contributions offer a powerful separation mechanism. In most instances, R & S enantiomers separated on the quinine form (QN-1) of the column can be eluted in reverse order when using the quinidine (QD-1) column (See Fig 12). The complementary separation thus obtained can be a powerful tool when scaling up the separation for purification of a given enantiomer. High Loadability Chiral AX columns offer excellent loadability, up to 50 mg of sample per gram of stationary phase. This high capacity coupled with the complementary separations obtained makes ProntoSIL Chiral AX columns in the QN-1 and QD-1 phases an excellent preparative system for chiral compounds (Fig 13). Figure 13 ProntoSIL Chiral AX Columns Show Extremely High Loadability Analytical 40 mg injected Column: ProntoSIL Chiral AX QN x 150 mm Mobile Phase: 10 mm ammonium acetate + 30 mm acetic acid in Methanol, ph = 6 Flow Rate: 1 ml/min Temperature: 25 C Time (min) Literature References Quinine and Quinidine Derivatives as Chiral Selectors I. Brush Type Chiral Stationary Phases for HPLC Based on Cinchonan Carbamates and Their Application as Chiral Anion Exchangers, M.Lämmerhofer and W.Lindner, J.Chromatogr. A 741, (1996). Chiral Anion Exchange-Type Stationary Phases Based on Cinchonan Alkaloids. An effective tool for the separation of the enantiomers of chiral acids. M. Lämmerhofer, N.M.Maier and W. Lindner, American Laboratory 30/8, (1998). Enantioselective Anion Exchangers Based on Cinchona Alkaloid Derived Carbamates: Influence of C8/C9-Stereochemistry on Chiral Recognition, N.M.Maier, L. Nicoletti, M.Lämmerhofer and W. Lindner, Chirality 11, (1999). Enantiomeric Separation of N-protected Amino Acids by Nonaqueous Capillary Electrophoresis Using Tert.Butyl Carbamoylated Quinine as Chiral Additive, V. Piette, M. Lämmerhofer,W. Lindner, J.Crommen, Chirality 11, (1999). ProntoSIL Chiral AX Ordering Information Dimensions Particle (mm) Size (μm) AX QD-1 AX QN x F10EPS x F10FPS x F10EPS x F10FPS x F10EPS x F10FPS050 Guard Cartridges 2.0 x 10 (3 pk) F10FPS F10EPS x 10 (3 pk) F10FPS F10EPS05 Guard Cartridge Holder ProntoSIL Chiral AX columns are also available in preparative dimensions

12 HPLC Columns ProntoSIL HPLC Column Application - 1 Contents Cold medicine Phenol mixture EPA 604/625 Tricyclic Antidepressants I Fat soluble Vitamins II Engelhardt test for RP phases Antirheumatic bath ingredient High Speed Separation of Catecholamines Organic Acids I Organic Acids II Formic Acid/Acetic Acid Water soluble Vitamins Peptides I Proteins I Fat soluble Vitamins I Insulin Derivatives I Insulin Derivatives II Isomers of Tocopherol (Vitamin E) Tricyclic Antidepressants II Steroids - UV vs. Evaporative Light Scattering Detection (ELSD) Carotenoids I N-Methylated Xanthines Parabens Carbohydrates I Carbohydrates II Evaporative Light Scattering Detection Amino Acids I Weak Anions - Ion Pair Chromatography Iodate in Galvanic Bath Proteins II Peroxide 5-Aminolevulinic Acid Alcohols I Dihydroxybenzoic Acids Bitter ingredients of hop I Bitter ingredients of hop II Melatonin Aldehydes and Ketones I: DNPH Derivatives in Galvanic Bath Fast Separation of Proteins Fast Separation of Peptides Bischoff

13 FUTECS your chromatography specialist Cold medicine ProntoSIL Eurobond C18 5 µm Phenol mixture EPA 604/625 ProntoSIL EnviroPHE Part number 1204F181PS050 Dimension 125 x 4.0 mm Eluent A: 5mM Li 2 SO 4 in H 2 O4; ph 2.1 B:ACN/50mM H 3 PO 4 Gradient 0%B, s; 0_12% B, s; 12-17% B, s; 17-39% B, s Flow 1 ml/min Detection UV 230nm, s; UV 261nm, s Temperature 20 Injection 3 µl 1 : Ascorbic acid 2 : Paracetamol 3 : Coffein 4 : Chlorphenamin Part number 1246F440PS30 Dimension 125 x 4.6 mm Eluent A: H 2 O/1% HAc B: MeOH/1% HAc Gradient 5-100% B, 0-30min Flow 1 ml/min Detection UV 280 nm Temperature 25 Injection 2 µl Supelco Calibration Standard (P/N: ) 1: Phenol 2: 4-Nitrophenol 3: 2.4-Dinitrophenol 4: 2-Chlorophenol 5: 2-Nitrophenol 6: 2.4-Dinmethylphenol 7: 4.6-Dinitro-o-Cresol 8: 2.4-Dichlorophenol 9: 4-Chloro-m-Cresol 10: Trichlorophenol 11: Pentachlorophenol Tricyclic Antidepressants I ProntoSIL CN Fat soluble Vitamins II ProntoSIL C18 SH Part number 2546F200PS050 Dimension 250 x 4.6 mm Eluent A: 25mM K 2 HPO 4 (ph 7.1) B: MeOH/ACN (15/65) 25% A, 75% B Flow 1 ml/min Detection UV 254nm Temperature 40 Concentration 50 ppm each 1 : Trimipramine 2 : Doxepin 3 : Amitriptyline 4 : Imipramine 5 : Nortriptyline 6 : Desipramine 7 : Protriptyline Part number Dimension Eluent Flow Detection 2503F180PS x 3.0 mm MeOH 1 ml/min Evap. Light Scattering Detector (DDL 31) PMT: 600, T: 33 Temperature 20 Concentration ppm each 1 : Vitamin A 2 : Vitamin A acetate 3 : Vitamin D 2 4 : Vitamin D 3 5 : Vitamin E 6 : Vitamin K

14 HPLC Columns Engelhardt test for RP phases ProntoSIL Eurobond C18 5 µm Antirheumatic bath ingredient ProntoSIL Eurobond C18 5 µm Part number 1204F181PS050 Dimension 125 x 4 mm Nr. Ret.(min) TP TP/m Asym. Name Uracil Eluent H 2 O/MeOH 51/49 (w/w) Aniline Flow 1 ml/min Phenol p-ethylaniline Detection UV 254nm N.N-Dimethylaniline Temperature Toluene Ethylbenzene Part number Dimension Eluent Flow Detection 2504F181PS x 4 mm H 2 O/MeOH 30/70 (w/w). 0.1% acetic acid 0.9 ml/min UV 254nm Temperature 20 Injection 20 µl 1 : Camphor 2 : Nicotinic acid methyl easter 3 : Nicotinic acid benzyl easter 4 : Ethylenegly colmonosalicylate High Speed Separation of Catecholamines Organic Acids I ProntoSIL C18 AQ ProntoSIL C18 AQ Part number 0404F184PS030 Dimension 33 x 4 mm Eluent CAT-A-Phase Flow 1.0 ml/min Detection Coulometric Temperature 30 1 : Norepinephrine 2 : Epinephrine 3 : Dihydroxybenzylamine 4 : Dopamine Part number 2503F184PS030 Dimension 250 x 3.0 mm Eluent 50 mm H 3 PH 4 Flow 0.7 ml/min Detection UV 205nm Temperature 22 1 : Glutamic acid 2 : Oxalic acid 3 : Tartaric acid 4 : Malic acid 5 : Ascorbic acid 6 : Acetic acid 7 : Maleic acid 8 : Citric acid 9 : Fumaric acid ProntoSIL C18 AQ Part number 0203F184PS030 Dimension 25 x 3 mm Eluent ADAM-B-Phase Flow 0.95 ml/min Detection Coulometric Temperature 28 Injection 1 µl 1 : Norepinephrine 2 : Epinephrine 3 : Dihydroxybenzylamine 4 : Dopamine 208 Bischoff

15 FUTECS your chromatography specialist Organic Acids II ProntoSIL C18 AQ Formic Acid / Acetic Acid ProntoSIL C18 AQ Part number 2003F184PS030 Dimension 300 x 3.0 mm Eluent A: H 2 O/ 50 mm H 3 PO 4 B: ACN/ 50mM H 3 PO 4 Gradient 0-100% B, 0-20 min Flow 0.6 ml/min Detection UV 215 nm Temperature 25 1 : Oxalic acid 2 : Tartaric acid 3 : Pyridine-4-carboxylic acid 4 : Pyridine-3-carboxylic acid 5 : Lactic Acid 6 : Citric Acid 7 : Pyridine-2,6-dicarboxylic acid 8 : 4-hydroxybenzoic acid 9 : 3-Hydroxybenzoic acid 10 : Benzoic acid 11 : 2-Hydroxybenzoic acid (= Salicylic acid) Part number 2503F184PS030 Dimension 250 x 3.0 mm Eluent 100 mm H 3 PO 4 Flow 0.7 ml/min Detection UV 202 nm Temperature 22 1 : Formic acid 2 : Acetic acid Water soluble Vitamins ProntoSIL C18 AQ Peptides I ProntoSIL C18 H Part number 2003F184PS030 Dimension 200 x 3.0 mm Eluent A: H 2 O/ 50 mm H 3 PO 4 B: ACN Gradient 0% B, s; 0-30% B, s; 30% B, s Flow 0.7 ml/min Detection UV 230 nm Temperature 22 1 : Pyridoxamine 2 : Thiamine (Vit. B1)+impurity 3 : Ascorbic acid (Vit. C) 4 : Nicotinic acid (Niacin) 5 : Nicotinamide (Vit. B 3 ) 6 : Pyridoxal 7 : Pyridoxine (Vit. B 6 )+ impurity of Vit. B 12 8 : Folic acid 9 : Cyanocobalamin (Vit. B 12 ) 10 : Riboflavin (Vit. B 2 ) Part number 2546K185PS050 Dimension 250 x 4.6 mm Eluent Gradient Flow Detection A: H 2 O/ 0.1% TFA B: ACN/ H 2 O, 70/30 (v/v) 0.1%TFA % B, 0-30 min 1.5ml/min UV 220 nm Temperature 30 1 : Oxytocin 2 : Bradykinin 3 : Angiotensin II 4 : Eledoisin 5 : Neurotensin 6 : Angiotensin I 209

16 HPLC Columns Proteins I ProntoSIL C18 H Fat soluble Vitamins I ProntoSIL C18 H Part number 2546K185PS050 Dimension 250 x 4.6 mm Eluent A: H 2 O/ 0.1% TFA B: ACN/ H 2 O, 95/5 (v/v) 0.1% TFA Gradient 25-80% B, 0-17min Flow 1.5 ml/min Detection UV 220 nm Temperature 30 1 : Ribonuclease A 2 : Insulin, bovine 3 : Lysozyme 4 : BSA 5 : Myoglobin 6 : Ovalbumin Part number 2003F185PS030 Dimension 200 x 3.0 mm Eluent Acetonitrile Flow 1 ml/min Detection UV 280 nm Temperature 30 1 : Vitamin A 2 : Vitamin D2 3 : Vitamin D3 4 : α - Tocopherol Insulin Derivatives I ProntoSIL C18 H Insulin Derivatives II ProntoSIL C18 H Part number 2546K185PS050 Dimension 250 x 4.6 mm Eluent A: H 2 O/ 0.1% TFA B: ACN/ H 2 O, 95/5 (v/v) 0.1% TFA Gradient 25-80% B, 0-17min Flow 1.5 ml/min Detection UV 220 nm Temperature 30 1 : Protamine Sulfate 2 : Phenol 3 : Cresol 4 : Insulin H 5 : Protamine insulin Part number 2546K185PS050 Dimension 250 x 4.6 mm Eluent A: H 2 O/ 0.1% TFA B: ACN/ H 2 O, 95/5 (v/v) 0.1% TFA Gradient 25-80% B, 0-17min Flow 1.5 ml/min Detection UV 220 nm Temperature 30 1 : Cresol 2 : Insulin H 3 : Insulin Chain A, oxidized 4 : Insulin, bovine 5 : Insulin Chain B, oxidized 210 Bischoff

17 FUTECS your chromatography specialist Isomers of Tocopherol (Vitamin E) ProntoSIL C30 Tricyclic Antidepressants II ProntoSIL C18 AQ Part number 2546H300PS030 Dimension 250 x 4.6 mm Eluent MeOH/H 2 O, 96/4 (v/v) Flow 0.9 ml/min Detection Coulometric Temperature 25 1 : δ - Tocopherol 2 : γ - Tocopherol 3 : β - Tocopherol 4 : α - Tocopherol Part number 0604F184PS030 Dimension 53 x 4.0 mm Eluent ACN/MeOH/NH 4 H 2 PO 4 (10 mm), 62/13/23 (v/v), titration with TEA to ph 5 Flow 0.69 ml/min Detection UV 254 nm Temperature 20 1 : Doxepin 2 : Imipramine 3 : Nortriptyline 4 : Amitriptyline 5 : Trimipramine Steroids - UV vs. Evaporative Light Scattering Detection (ELSD) Carotenoids I ProntoSIL C30 ProntoSIL C18 H Part number 2003F185PS030 Dimension 200 x 3 mm Eluent MeOH Flow 0.65 ml/min Detection a) UV 200 nm b) ELSD: PMT gain: 600. T: 40 Temperature 30 Concentration 70 ppm each 1 : Hydrocortisone 2 : Progesterone 3 : Cholecalciferol 4 : Ergocalciferol 5 : Ergosterol 6 : Cholesterol Part number 2546H300PS030 Dimension 250 x 4.6 mm Eluent MeOH/ TBME, 80/20 (v/v) Flow 1.4 ml/min Detection VIS 450 nm Temperature 20 β- Carotene, technical mixture, isomerized 1 : all-trans 2 : 9-cis 211

18 HPLC Columns N-Methylated Xanthines ProntoSIL C18 AQ Parabens ProntoSIL PHENYL Part number 1504F184PS050 Dimension 150 x 4.0 mm Eluent 25mM NaH 2 PO 4, ph 3/ MeOH 80/20 (v/v) Gradient 25-80% B, 0-17min Flow 1.0 ml/min Detection UV 254 nm Temperature 35 Injection 10 µl 1 : Theobromine 2 : Theophylline 3 : Caffeine Part number 1204F050PS030 Dimension 125 x 4.0 mm Eluent 20mM K2HPO4 / ACN, 50/50 (v/v) Flow 1.0 ml/min Detection UV 254 nm Temperature 40 Injection 10 µl Parabens 1 : Methylparaben 2 : Ethylparaben 3 : Propylparaben 4 : Butylparaben Carbohydrates I ProntoSIL NH 2 Carbohydrates II Evaporative Light Scattering Detection ProntoSIL NH2 Part number 1246F190PS030 Dimension 125 x 4.0 mm Eluent H2O/ACN, 20/80 (v/v) Flow 1.0 ml/min Detection RI Temperature Ambient Injection 6 µl 1 : Fructose 2 : Mannose 3 : Glucose 4 : Galactose 5 : Saccharose 6 : Maltose Part number Dimension Eluent Flow Detection Temperature 1246F190PS x 4.0 mm H2O/ACN, 20/80 (v/v) 1.0 ml/min RI Ambient 1 : Fructose 2 : Mannose 3 : Glucose 4 : Galactose 5 : Saccharose 6 : Maltose Injection 6 µl 212 Bischoff

19 FUTECS your chromatography specialist Amino Acids I ProntoSIL C18 H Weak Anions - Ion Pair Chromatography ProntoSIL C8 SH Part number 2003F185PS030 Dimension 200 x 3.0 mm Eluent A: 20 mm CH 3 COONa in H 2 O/ACN, 93/3 (v/v) B: 20 mm CH 3 COONa in H 2 O/ACN, 50/50 (v/v) Gradient 5-28% B, s; 28-45% B, s 45-82% B, s 82-90% B, s Flow Detection Temperature 30 Injection 1 µl 0.6 ml/min UV 340 nm SIGMA standard AA-s- 18: OPA/ Mercaptopropionic acid labeled Part number 2003F080PS030 Dimension 200 x 3.0 mm Eluent 10mM KH 2 PO 4 /10 mm TBA-H 3 PO 4, ph 2.4 Flow 0.6 ml/min Detection UV 205 nm Temperature 25 1 : acetic acid 2 : lactic acid 3 : malic acid 4 : iodate 5 : citric acid Iodate in Galvanic Bath ProntoSIL C18 SH Proteins II ProntoSIL C4 Part number 2546F180PS050 Injection 20 µl Part number 2003K040PS030 Detection UV 220 nm Dimension 200 x 4.6 mm Eluent 10mM KH 2 PO 4 / 2 mm TBA-H 3 PO 4, ph 1.95 Flow 1 ml/min Detection UV 196 nm Temperature 25 Galvanic bath, technical 1 : iodate Dimension Eluent 200 x 3.0 mm A: H 2 O/0.1% TFA B: ACN/H2O, 95/5 (v/v) 0.1% TFA Gradient 10-40% B, s; 40-47% B, s; % B, s Flow 0.6 ml/min Temperature 30 1 : Cytochrome c 2 : Fibrinogen, human 3 : Chymotrypsinogen 4 : β-lactoglobulin 5 : Thyroglobulin 213

20 HPLC Columns Peroxide ProntoSIL C18 H 5-Aminolevulinic Acid ProntoSIL C18 H Part number 0603F185PS030 Dimension 53 x 3.0 mm Eluent Flow A: 35mM citric acid/ 85mM sodium acetate/ 0.5mM heptane sulphonate: ph 4.3 B: MeCH A/B: 99.7/0.3 (w/w) 0.5 ml/min Detection Coulometric Temperature 25 Concentration 500 ppb 1 : Perbenzoic acid t-butyl ester Part number 1204F185PS030 Dimension 125 x 4.6 mm Eluent H 2 O/MeOH/THF, 90/6/4 (v/v) 0.5 mm octane sulphonic acid (sodium salt): ph 3.26 Flow 1 ml/min Detection Conductivity; FS: 5 µs Temperature 26 Injection 10 µl Concentration 100 ppb 1 : 5-Aminolevulinic acid Alcohols I ProntoSIL C18 AQ Dihydroxybenzoic Acids ProntoSIL CN Part number 1546F184PS050 Dimension 150 x 4.6 mm Eluent H 2 O/MeOH, 96/4 (v/v) 0.8 mm Caps ; ph 2.5 Flow 1.1 ml/min Detection Indirect Conductivity ; FS: 500 µs Temperature 26 Injection 10 µl Concentration 10 ppb each 1 : Ethanol 2 : Isopropanol 3 : n-propanol Part number 2003F200PS030 Dimension 200 x 3 mm Eluent 30mM Sodium acetate/ 30mM Sodium Citrate; ph ml/min Flow Detection 0.5 ml/min UV 220 nm Temperature 20 Concentration 1.8 mmol each 1 : 2.3-Dihydroxybenzoic Acid 2 : 2.5-Dihydroxybenzoic Acid 3 : Salicylic Acid (1.2-DHBA) 214 Bischoff

21 FUTECS your chromatography specialist Bitter ingredients of hop I ProntoSIL C18 SH Bitter ingredients of hop II ProntoSIL C8 SH Part number 0604F180PS050 Dimension 53 x 4 mm Eluent H2O/MeOH 25/75 (v/v) + 50 mm H 3 PO 4 Flow 1 ml/min Detection UV 314 nm Temperature 21 Injection 10 µl Hop CO 2 -extract 1 : not identified 2 : Cohumulon 3 : Deoxyhumulon 4 : n-plus Adhumulon 5 : Colupulon 6 : n-plus Colupulon Part number 2504F080PS050 Dimension 250 x 4 mm Eluent H2O/MeOH 20/80 (v/v) + 50 mm H 3 PO 4 Flow 1 ml/min Detection UV 314 nm Temperature 21 Injection 10 µl Hop CO 2 -extract 1 : not identified 2 : Cohumulon 3 : Deoxyhumulon 4 : n-plus Adhumulon 5 : Colupulon 6 : n-plus Colupulon Melatonin ProntoSIL C18 H Aldehydes and Ketones I: DNPH Derivatives in Galvanic Bath ProntoSIL C18 H Part number 1003F185PS030 Dimension 100 x 3 mm Eluent ESA CAT-A Phase Flow 0.5 ml/min Detection Coulometric Temperature 25 Injection 10 µl concentration 10 ppb 1 : Melatronin Part number 2546F185PS050 Dimension 250 x 4.6 mm Eluent A : H2O; B: ACN Gradient 40% B, 0-60 s; 40-60% B, s; 60-80% B, s Flow 1 ml/min Detection UV 360 nm Temperature 30 Injection 50 µl concentration 10 ppb 1 : DNPH 2 : Formaldehyde 3 : Acetaldehyde 4 : Acetone 5 : Propanal 6 : Pentanal 215

22 HPLC Columns Fast Separation of Proteins ProntoSIL C18 H Fast Separation of Peptides ProntoSIL C18 H Part number 0746K185PS030 Detection Dimension 75 x 4.6 mm Eluent A: H 2 O/0.1% TFA B: ACN/ H 2 O 95/5 (v/v)/0.1% FTA Gradient a) 29-48% B, 0-25s; %B, s b) 29-48% B, 0-49 s; % B, s c) % B, s Flow a) 4ml/min; b) 2ml/min; c) 1ml/min Temperature 30 UV 220 nm 1: Ribouclease A 2: Insulin, bovine 3: Lysozyme 4: BSA 5: Myoglobin 6: Ovalbumin 7: not identified Part number 0746K185PS030 Dimension 75 x 4.6 mm Eluent A: H 2 O/0.1% TFA B: ACN/ H 2 O 70/30 (v/v), 0.1% FTA Gradient 35-80% B, s Flow 1 ml/min Detection UV 220 nm Temperature 30 1: Oxytocin 2: Bradykinin 3: Angiotensin I 4: Eledoisin 5: Neurotensin 6: Angiotension II 216 Bischoff

23 FUTECS your chromatography specialist ProntoSIL HPLC Column Application - 2 Contents Ketones l Organic Acids lll Coconat Fatty Acid - Ethoxylates 10 EO Castor oil Ethoxylates 20 EO Soybean oil - Ethoxylates 20 EO Calcium-DL-2-Hydroxy-4-methylthiobutyrate in Animal Food Ubiqinols and Ubiquinones Aldehydes and Ketones as DNPH Derivatives Tryptic digest of Casein Peptone Aflatoxines with KBr Derivatization Peptone Amino Acids with FMOC Chinin and Saccharin in Beverages Flavonoids in Red Wine Analysis of Nitro-PAH with Fluorescence Detection (I) Analysis of Nitro-PAH with Fluorescence Detection (II)

24 HPLC Columns Ketones I ProntoSIL C18 AQ Organic Acids III ProntoSIL C18 AQ Part number 0203F184PS030 Dimension 25 x 3 mm Eluent A: H 2 O B: ACN Gradient % B, 0-18 min Flow 0.35 ml/min Detection UV 270 nm Temperature 30 Injection 2 µl 1: Tetramethylketone 2: 2-Pentanone 3: 2-Hexanone 4: 2-Hexanone 5: 2-Octanone 6 2-Nonanone 7: 2-Decanone 8: 2-Undecanone 9: 2-Dodecanone 10: 2-Tridecanone 11: 2-Hexadecanone Part number 3003F184PS030 Dimension 300 x 3 mm Eluent 5 mm Li2So4/ H2SO4 ph 2.81 Flow 0.56 ml/min Detection RI Temperature 20 Injection 10 µl Concentration 300 ppm each, except 1,3 1: oxalic acid 150 ppm 2: not identified 3: tartaric acid 150 ppm 4: quinic acid 5: malic acid 6: malonic acid 7: shikimic acid 8: lactic acid 9: ascorbic acid 10: acetic acid 11: citric acid 12: fumaric acid 13: succinic acid Coconat Fatty Acid Ethoxylates 10 EO ProtoSIL NH2 Castor oil Ethoxylates 20 EO ProtoSIL NH2 Part number 2546F190PS050 Dimension 250 x 4.6 mm Eluent A: n-hexane B: acetone Gradient 3-20% B, s; 20-38% B, s; 38-70% B, s 70% B, s Flow Detection Temperature 30 Injection 50 µl 1 ml/min Evap. Light Scattering Detector (ELSD), model DDl 31; PMT: 300, T: 42, p(air): 1bar Part number 2546F190PS050 Dimension 250 x 4.6 mm Eluent A: n-hexane B: acetone Gradient 0-5% B, s; 5-40% B, s 40% B, s Flow 1 ml/min Detection Evap. Light Scattering Detector (ELSD), model DDl 31; PMT: 500, T: 42, p(air): 1bar Temperature 30 Injection 50 µl 218 Bischoff

25 FUTECS your chromatography specialist Soybean oil Ethoxylates 20 EO ProtoSIL NH2 Part number 2546F190PS050 Dimension 250 x 4.6 mm Eluent A: n-hexane B: acetone Gradient 3-20% B, s; 20-38% B, s; 38-70% B, s 70% B, s Flow Detection Temperature 30 Injection 50 µl 1 ml/min Evap. Light Scattering Detector (ELSD), model DDl 31; PMT: 300, T: 42, p(air): 1bar Calcium-DL-2-Hydroxy-4- methylthiobutyrate in Animal Food ProtoSIL NH2 Part number 1003F185PS030 Flow 0.55 ml/min Dimension 100 x 3 mm Detection Coulometric Eluent A: 75 mm NaH2PO4/ 0.2 mm heptanesulfonic acid (sodium salt), ph 2.9 B: ACN; A/B 98/2 (v/v) Temperature Injection µl Ubiqinols and Ubiquinones ProtoSIL C18 SH Aldehydes and Ketones as DNPH Derivatives ProntoSIL C18 H Part number 12P4F180PS030 Dimension 125 x 4 mm PEEK Eluent MeOH/EtOH/i-Propanol 900/180/100(v/v) + COONH 4 (2g/l) Flow 1 ml/min Detection Coulometric Temperature 26 Injection 50 µl 1: δ-tocopherol 2: γ-tocopherol 3: β-tocopherol 4: Q9-OH 5: Q10-OH 6: α-carotene 7: β-carotene 8: Q9 9: Q10 Part 2504F185PS050 number Dimension 250 x 4.0 mm Eluent/Gradient (final compositions of linear steps are given (v/v/v ratios)): 0-5 min isocratic: THE/ACN/H 2 O (16/25/59) 10 min. The/ACN/H 2 O (10/40/50) 20 min. The/ACN/H 2 O (0/60/40) 30 min. The/ACN/H 2 O (0/80/20) 40 min. The/ACN/H 2 O (0/100/0) 45 min. The/ACN/H 2 O (16/25/59) Flow 1.3 ml/min Detection UV 360 nm Temperature 30 Injection 10 µl 1: Nonanal (internal standard) 2: 2,4-Dinitrophenylhydrazine 3: Formaldehyde 4: Acetalderhyde 5: Acetone 6: Acrolein 7: Propanal 8: Crotonic Aldehyde 9: n-butanal 10: Benzaldehyde 11: Pentanal 12: 2-Methylbenzaldehyde 13: 4-Methylbenzaldehyde 14: 3,3-Dimethylbutanone 15: Hexanal 16: 2,4-Dimethylbenzaldehyde 17: 2,4-dimethylpentanone 18: Heptanal 19: Octanal 219

26 HPLC Columns Tryptic digest of Casein Peptone ProntoSIL C18-H Aflatoxines with KBr Derivatization ProntoSIL C18 AQ Part number 0746K180PS030 Dimension 75 x 4.6 mm Eluent A: H 2 O/0.1% TFA B: ACN/ H 2 O 70/30 (v/v), 0.1% TFA Gradient 0-40% B, s 40-0% B, s Flow 1 ml/min Detection UV 220 nm Temperature 30 Tryptic digest of casein peptone Part number 2546F184PS050 Dimension 250 x 4.6 mm Eluent ACN/MeOH/H2O 20/20/60 (v/v/v) Flow 1.0 ml/min Detection Fluorescence Detector Model 8470 Ex: 365nm Em:429nm Temperature 30 Aflatoxin-Standard G2: 60 fg/ml G1: 100 fg/ml B2: 50 fg/ml B1: 90 fg/ml Injection: 100 µl Amino Acids with FMOC ProntoSIL-AA-FMOC 5.0µm Chinin and Saccharin in Beverages ProntoSIL C18 H 5.0 µm Part number 2504F470PS050 Dimension 250 x 4.0 mm Eluent A: 50 mm CH 3 COONa ph 4.2, 5% THF B: ACN Flow 1.0 ml/min Gradient 20-36% B, 0-20 min 36-38% B, min 38% B, min 38-59% B, min 59-67% B, min Detection Fluorescence Ex: 263 nm Em: 313 nm Temperature 30 Injection 10 µl SIGMA standard AA-S Asn, Gln, IS (ß-tAla) FMOC labeled Part number 1204F185PS050 Dimension 125 x 4.0 mm Eluent MeOH/H 2 O 70/30 (v/v) + 5ml/l H3PO4 Flow 1.0 ml/min Detection UV 210 nm Temperature 25 Injection 20 µl Bitter Lemon Light 1: Quinine 2: Saccharin 220 Bischoff

27 FUTECS your chromatography specialist Flavonoids in Red Wine ProntoSIL C8 ace-eps 5.0µm Analysis of Nitro-PAH with Fluorescence Detection (I) ProntoSIL Phenyl Part number 1204F08APS050 Dimension 125 x 4.0 mm Eluent A: 10 mm NaH 2 PO 4 ph 3.0 B: ACN Flow 2.5 ml/min Gradient Gradient: 30% B, 0-2 min 30-70% B, 2-4 min 70% B, min 70-30% B, min 30% B, min Detection UV 280 nm (Stilbenes) 370 nm (Flavonoides) Temperature 40 Injection 25 µl Red Wine 1: Myrcetin 2: cis- Resveratrol 3: trans- Resveratrol 4: Quercetin 5: Caempferol Part number 1204F050PS030 Dimension 125 x 4.0 mm Eluent A: MeOH/H 2 O 60/40 (v/v) B: MeOH 0 % B hold for 13 min, % B in 29 min Flow 0.8 ml/min Detection Fluorescence Detector Model 8470 wavelength program Temperature 25 Injection 20 µl Nitro-PAH Mix1 Analysis of Nitro-PAH with Fluorescence Detection (II) ProntoSIL Phenyl Part number 1204F050PS030 Dimension 125 x 4.0 mm Eluent A: MeOH/H 2 O 60/40 (v/v) B: MeOH 0 % B hold for 13 min, % B in 29 min Flow 0.8 ml/min Detection Fluorescence Detector Model 8470 wavelength program Temperature 25 Injection 20 µl Nitro-PAH Mix

28 HPLC Columns ProntoSIL HPLC Column Application - 3 Contents Carbohydrates, Organic Acids and Alcohols in Wine Dimethylsiloxane with Evaporative Light Scattering Detection Fast Analysis of a pharmaceutical Teststandard with Multiwavelength Detection Fast Analysis of a Cold Medicine II with Multiwavelength Detection Standardtestmixture with Multiwavelength Detection Fast Analysis of Parabens II with Multiwavelength Detection Fat soluble Vitamins II with Multiwavelength Detection Aromatic Hydrocarbons according to pren Diesel Acetaldehyde in Mineralwater Ergosterol in Grass Sulfa Drugs Bischoff

29 FUTECS your chromatography specialist Carbohydrates, Organic Acids and Alcohols in Wine Carbohydrate H+ Dimethylsiloxane with Evaporative Light Scattering Detection ProntoSIL C18 SH Part number Dimension 300 x 7.8 mm Eluent 1.25 mm H 2 SO 4 Flow 0.6 ml/min Detection RI Temperature 45 Injection 50 µl Wine Complett Standard Part number 2546F180PS050 Detection Dimension 250 x 4.6 mm Eluent A: ACN/Acetone 70/30 (v/v) B: Ethyl acetate 5-70% B in 50 min, 70% B hold Temperature 25 for 15 min Flow 0.9 ml/min Evap. Light Scattering detector (DDL31) PMT: 500, Temp: 40 Pressure (air): 1.5 bar Silicone-Standard (DMS-T11) Injection: 10 µl Fast Analysis of a pharmaceutical Teststandard with Multiwavelength Detection ProntoSIL C18 AQ Fast Analysis of a Cold Medicine II with Multiwavelength Detection ProntoSIL C18 AQ Part number Dimension Eluent Gradient Flow Detection 0446F184PS x 4.6 mm A: H 2 O + 0.1% TFA; B:ACN 20-30% B in 60 sec 1.2 ml/min Multiwavelength Detector (DAD-3L) Temperature 25 Injection 1 µl 1 Ascorbinic acid 2 Paracetamol 3 Caffeine 4 Acetylsalicylic acid Part number Dimension Eluent Gradient Flow Detection 0446F184PS x 4.6 mm A: H 2 O + 0.1% TFA; B:ACN 20-30% B in 30 sec 1.2 ml/min Multiwavelength Detector (DAD-3L) Temperature 25 Injection 1 µl 1 Paracetamol 3 Caffeine 4 Propylphenanzone Wavelength 230, 261 and 274 nm; Ref. at 350nm Wavelength 230, 254 and 274 nm; Ref. at 350nm 223

30 HPLC Columns Standardtestmixture with Multiwavelength Detection ProntoSIL C18 SH Fast Analysis of Parabens II with Multiwavelength Detection ProntoSIL C18 AQ Part number 0604F180PS030 Dimension 53 x 4.0 mm Eluent H2O/ ACN; 40/60 Flow 1.0 ml/min Detection Multiwavelength Detector (DAD-3L) Wavelengthe 254, 262 and 273 nm Ref. at 350nm Temperature 25 Injection 1 µl 1 Acetone 2 Benzene 3 Toluene 4 m-xylene Part number 0446F184PS050 Dimension 33 x 4.6 mm Eluent A: H2O B: ACN Gradient 20-30% B in 30 sec Flow 1.0 ml/min Detection Multiwavelength Detector (DAD-3L) Wavelengthe 210, 230 and 254 nm Ref. at 350 nm Temperature 25 Injection 2 µl 1 Uracil 2 Methylparabene 3 Ethylparabene 4 Propylparabene Fat soluble Vitamins II with Multiwavelength Detection ProntoSIL C18 SH Aromatic Hydrocarbons according to pren ProntoSIL ENVIRO PTL Part number 2503F180PS030 Dimension 250 x 3.0 mm Eluent MeOH Flow 1 ml/min Detection Multiwavelength Detector (DAD-3L) Wavelengthe 225, 264, 325 nm Temperature 25 1: Vitamin A 2: Vitamin A acetate 3: Vitamin D 2 4: Vitamin D 3 5: Vitamin E 6: Vitamin K 1 Part number 2546C450PS050 Dimension 250 x 4.6 mm Eluent Heptane Flow 1.0 ml/min Backflush on 16 min Detection RI Temperature 25 Injection 10 µl Calibration Standard B - EUOPEAN STANDARD pren (June 1997) 1: Cyclohexane (2.0g/100ml) 2: o-xylene (1.0g/100ml) 3: 1-Methylnaphthalene (1.0g/100ml) 4: Phenanthrene (0.2g/100ml) between Cyclohexane and o- Xylene (the resolution between Cyclohexane and o-xylene should not be less thean 5.0) 224 Bischoff

31 FUTECS your chromatography specialist Diesel ProntoSIL ENVIRO PTL Acetaldehyde in Mineralwater ProntoSIL C18 ACE- EPS Part number 2546C450PS050 Dimension 250 x 4.6 mm Eluent Heptane Flow 1.0 ml/min backflush on 20 min Detection RI Temperature 25 Injection 10 µl 1: Cyclohexane 2: Monocycl. Aromatics 3: Dicycl. Aromatics 4: Dicycl. Aromatics 5: Polycycl. Aromatics Part number 1504F18APS030 Dimension 150 x 4.0 mm Eluent H2O/ ACN 50/50 (v/v) Flow 1.0 ml/min Detection UV 360 nm Temperature 25 Injection 500 µl ProntoSIL HPLC Column Application - 4 Mineralwater after Derivatisation with DNPH 1 DNPH 5 Acetaldehyde. 6ppb Ergosterol in Grass ProntoSIL C18 H Sulfa Drugs ProntoSIL C18 ACE - EPS Part number 1204F185PS050 Dimension 125 x 4.0 mm Eluent H2O/MeOH 2/98 (v/v) Flow 0.75 ml/min Detection UV 282 nm Temperature 25 Injection 20 µl 1 Ergosterol Extracted according to VDLUFA (1993) Part number 1546F18APS050 Dimension 150 x 4.6 mm Eluent MeOH/H2O/Acetic Acid 20/79/1 (v/v/v) Flow 1 ml/min Detection UV 254 nm Temperature 25 Injection 3 µl 1 Sulfanilamide 2 Sulfadiazine 3 Sulfathiazole 4 Sulfamerazine 5 Sulfamethazine 225

32 HPLC Columns ProntoSIL HPLC Column Application - 4 Contents Sugaralcohols, Mono-and Disaccharides with BioQuant Carbohydrate Inositol Alcohol, Sugaralcohols, Mono-and Disaccharides with BioQuant Carbohydrate 226 Sugaralchols, Mono-and Disaccharides with BioQuant Carbohydrate Alcohol, Sugaralcohols, Mono-and Disaccharides with BioQuant Carbohydrate Column Sugar1 Dimension 250 x 4.6 mm PEEK Eluent 100mM NaOH Flow 0.9 ml/min Detection BioQuant Carbohydrate Temperature 28 Injection 20 µl 1 Inositol 2 Arabitol 3 Sorbitol 4 Fucose 5 Arabinose 6 Glucose 7 Xylose 8 Fructose 9 Lactose 10 Sucrose (1-50 mg/l) Column Dimension Eluent Gradient Flow Detection Temperature 28 Injection 20 µl Sugar1 250 x 4.6 mm PEEK A: 96mM NaOH B: 96mM NaOH, 250 mm NaOAc 5%-10%B 0-15min; 10-25%B min 1 ml/min BioQuant Carbohydrate 1 Ethanol 2 Inositol 3 Arabitol 4 Sorbitol 5 Fucose 6 Arabinose 7 Glucose 8 Xylose 9 Fructose 10 Lactose 11 Sucrose 12 Maltose ( mg/l) 226 Bischoff

33 FUTECS your chromatography specialist ProntoSIL HPLC Column Application - 5 Contents High Speed Separation of Catecholamines 227 High Speed Separation of Catecholamines ProntoSIL C18 AQ High Speed Separation of Catecholamines ProntoSIL C18 AQ Part number Dimension Eluent Flow 0404F184PS x 4 mm CAT-A-Phase 1.0 ml/min 1: Norepinephrine 2: Epinephrine 3: Dihydroxybenzylamine 4: Dopamine Part number Dimension Eluent Flow 0203F184PS x 3 mm ADAM-B-Phase 0.95 ml/min 1: Norepinephrine 2: Epinephrine 3: Dihydroxybenzylamine 4: Dopamine Detection Coulometric Detection Coulometric Temperature 30 Injection 3 µl Temperature 28 Injection 1 µl 227

34 HPLC Columns ProntoSIL HPLC Column Application - 6 Contents Sugaralcohols, Mono-and Disaccharides with BioQuant Carbohydrate Sugars in Applejuice with BioQuant Carbohydrate Sugars in Applejuice Sugars in Orangejuice (A) and Diet Orangejuice (B) Sugars in Shampoo Maltooligosaccacharides from Cornsyrup Monosaccharides of Glycoproteins Plasma Catecholamines Catecholamine Standard Catecholamine Standard Bischoff

35 FUTECS your chromatography specialist Sugaralcohols, Mono-and Disaccharides with BioQuant Carbohydrate Sugars in Applejuice with BioQuant Carbohydrate Column PAD Column Dimension 250 x 4.6 mm PEEK Eluent 100mM NaOH Flow 0.9 ml/min Detection BioQuant Carbohydrate Temperature 28 Injection 20 µl 1 Inositol 2 Arabitol 3 Sorbitol 4 Fucose 5 Arabinose 6 Glucose 7 Xylose 8 Fructose 9 Lactose 10 Sucrose (1-50mg/l) Column PAD Column Dimension 250 x 4.6 mm PEEK Eluent 100 mm NaOH Flow 0.9 ml/min Detection BioQuant carbohydrate Temperature 28 Injection 20 µl (diluted 1: 1000) 1 Sorbitol 2 Glucose 3 Fructose 4 Sucrose Sugars in Applejuice Sugars in Oragnejuice (A) and Diet Orangejuice (B) Column Dimension Eluent Flow hamilton RCX x 4.6 mm PEEK 60 mm NaOH 2.00 ml/min 1 Sorbitol 2 Glucose 3 Fructose 4 Sucrose Column Dimension Eluent Flow Hamilton RCX x 4.6 mm PEEK 60 mm NaOH 2.00 ml/min (diluted 1: 1000) 1 Glucose 2 Fructose 3 Sucrose Detection BioQuant Carbohydrate Detection BioQuant carbohydrate Temperature 32 Injection 1 µl (diluted 1:1000) Temperature

36 HPLC Columns Sugars In Shampoo Maltooligosaccacharides from Cornsyrup Column Hamilton RCX-30 Dimension 150 x 4.6 mm PEEK Eluent 60 mm NaOH Flow 2.00 ml/min Detection BioQuant Carbohydrate Temperature 32 Injection 1 µl 3 Sorbitol 5 Glucose 6 Fructose 7 Sucrose Column Dimension Eluent Gradient Hamilton RCX x 4.6 mm PEEK Eluent A: 60 mm NaOH Eluent B: 60 mm NaOH: 500 mm Na-acetat 10-60% B in 0-20 min Flow 2.00 ml/min Detection BioQuant carbohydrate Temperature 32 G4- G10 Monosaccharides of Glycoproteins Plasma Catacholamines Column Dionex CarboPAC PA1 Column ProntoSIL C18 AQ Dimension 250 x 4.0 mm PEEK Eluent 20 mm NaOH Flow 1.00 ml/min Detection BioQuant Carbohydrate Temperature 28 1 Galactosamine 2 Glucosamine Dimension 33 x 3.0 mm Eluent Pronto- Cat Phase Flow 0.5 ml/min Detection BioQuant carbohydrate Temperature 30 1 Noradrenalin 2 Adrenalin 3 DHBA 4 Dopamin 230 Bischoff

37 FUTECS your chromatography specialist Catecholamine -6 -Standard Catecholamine -8 -Standard Column ProntoSIL C18 AQ Dimension 33 x 3.0 mm Eluent Pronto- Cat Phase Flow 0.5 ml/min Detection BioQuant Carbohydrate Temperature 30 1 Arenalin 2 DHBA 3 DOPAC 4 5-HIAA 5 HVA 6 Serotonin Column ProntoSIL C18 AQ Dimension 33 x 3.0 mm 1 Noradrenalin Eluent Pronto- Cat Phase 2 Adrenalin Flow 0.5 ml/min 3 DHBA 4 DOPAC Detection BioQuant CAT Amine, +500 mv 5 Dopamin Temperature HIAA 7 HVA 8 Serotonin 231

38 HPLC Columns ProntoSIL Column 종류와특성 Contents ProntoSIL C18(C8) ace-eps Columns(60A, 120A, 200A, 300A) 233 ProntoSIL C18 H Columns(60A, 120A, 200A, 300A) 235 ProntoSIL C18(C8) SH Columns(60A, 120A, 200A, 300A) 237 ProntoSIL C18 AQ Columns(60A, 120A, 200A, 300A) 239 ProntoSIL Enviro C18 Columns(5um, 6um, 7um) ProntoSIL Hypersorb ODS Columns(3um, 5um) 241 ProntoSIL Spheribond ODS Columns(3um, 5um, 10um) ProntoSIL Eurobond C18 Columns(5um) 242 ProntoSIL C30 Columns(3um, 5um, 10um) ProntoSIL AMINO Columns(3um, 5um, 10um) 243 ProntoSIL Silica Columns(3um, 5um) 245 ProntoSIL Phenyl Columns(3um, 5um, 10um) 247 ProntoSIL CN Columns(3um, 5um, 10um) 248 ProntoSIL DIOL Columns(3um, 5um, 10um) Bischoff