ColumnsAcclaim PolarAdvantage II (PA2) HPLC Columns

Transcription

1 ColumnsAcclaim PolarAdvantage II (PA) HPLC Columns A Unique and Complementary Alternative to Conventional C8.8 Column: Acclaim PA, µm Peaks: ( ppm each) Dimensions:. mm. Maleate Mobile Phase: MeOH/.% NH OH, ph, v/v, /. Labetalol Temperature: C. Metaraminol Flow Rate:. ml/min. Atenolol. Acebutolol Detection: UV, nm. Metoprolol. Timolol 8. Oxprenolol 8 A new line of silica-based polarembedded columns for pharmaceutical, environmental, chemical, food, and life science applications Separates polar and nonpolar compounds Exceptional hydrolytic stability (ph. ) High polarity for complementary selectivity to C8 columns Compatible with % aqueous mobile phases Good peak shapes for both acidic and basic compounds High column efficiency Good batch-to-batch reproducibility Broad range of applications Separates Polar and Nonpolar Compounds The separation of polar and nonpolar compounds in a single run on a reversed-phase column is often a difficult proposition for chromatographers because the polar compounds are eluted either in the void, or unresolved from one another at the beginning of the chromatogram. The challenge with reversed-phase C8 columns, for this type of application, is low retention of polar compounds under the conditions necessary to approximate a separation, dewetting in % aqueous mobile phases, and more or less the same selectivity regardless of the column vendor. Existing polar-embedded phases offer different selectivity from conventional C8 columns and symmetrical peak shapes for basic compounds, but often at the expense of hydrophobic retention and hydrolytic stability at both low and high ph. The Acclaim PA column is a novel, high-efficiency, silica-based, polar-embedded column, manufactured by bonding a specially designed proprietary amide-embedded ligand to - or -µm-diameter high-purity spherical silica. This column is compatible with % aqueous environments over a wide range of ph (see beta blockers in figure above), exhibits high polarity for selectivity complementary to conventional C8 columns, and provides excellent peak shapes and efficiencies for both basic and acidic compounds.

2 Exceptional Hydrolytic Stability Polar-embedded silica stationary phases provide several advantages over general-purpose C8 phases, including improved peak shapes of basic analytes, compatibility with highly aqueous mobile phases, and unique selectivity characteristics. However, the hydrolytic stability of polar-embedded columns is typically inferior to that of hydrophobic C8 supports, due to lower ligand coverage and the hydrophilic nature of the embedded group. To address the inferior hydrolytic stability issue, the scientists at Dionex have developed a polar-embedded column with enhanced hydrolytic stability that provides selectivity complementary to conventional C8 columns throughout the whole operating ph range (ph. to ). HPLC separations of polar analytes are often run under acidic conditions (<.) to reduce tailing of amine-containing compounds. While the acidic conditions improve peak shape, the harsh environment will shorten column life due to cleavage of the bonded phase, resulting in frequent column replacement and instrument downtime. Figure shows an accelerated low-ph degradation test using % TFA at ph. After days under these conditions, the Acclaim PA phase is stable, while the Discovery RP-Amide C and the SymmetryShield C8 columns show significant loss of the bonded phase. Figure shows a similar accelerated degradation test at high ph, using triethylamine at ph. to age the column. After h, the Acclaim PA column still retains 9% of its retention capacity, whereas the other commercially available columns have dropped to less than 8%, and in some cases less than %. Thus, as shown by Figures and, the proprietary bonding of the Acclaim PA column is capable of resisting hydrolytic attack by protecting the bonded phase at both low and high ph. % Remaining Retention 9 8 Time of Purge (Days) Figure. Accelerated stability test at low ph. % Remaining Efficiency 8 Acclaim PA Figure. Accelerated stability test at high ph. Polarity = [K BBA K phenol /K toluene ] x ZORBAX Bonus-RP SymmetryShield C8 Discovery RP-Amide RP- 8 Time of Purge (hours) 8 Acclaim C8 Luna() C8 Luna () C8 Acclaim PA ZORBAX Extend-C8 ZORBAX Bonus-RP Acclaim PA SymmetryShield C8 8 Discovery RP-Amide C ProntoSIL EPS C8 Column: µm Dimensions:. mm Mobile Phase: CH CN/% TFA*, ph v/v / Temperature: C Flow Rate:. ml/min Detection: UV, nm Analyte: Toluene *Trifluoroacetic acid Column: µm Flow Rate: ml/min Dimensions:. mm Analyte: Phenanthrene Detection: UV, Mobile Phase: Aged in CH CN/ mm triethylamine, ph. v/v / at C Tested in CH CN/H O v/v / at C. 9 Acclaim PA 8 ZORBAX Bonus-RP Figure. Polarity comparison.

3 High Polarity for Selectivity Complementary to C8 Columns The ability of a reversed-phase column to retain polar compounds can be estimated based on the polarity of the stationary phase. The more polar the stationary phase, the longer the polar compounds will be retained. Figure shows a comparison of the polarity of a series of commercially available conventional C8 columns and polarembedded columns. The Acclaim C8 column and the Luna () C8 are representative of conventional C8 columns; the other columns are polarembedded. As expected, the conventional reversed-phase columns exhibit lower polarity than the polar-embedded phases. Included in Figure are three of the Acclaim family of columns from Dionex, the Acclaim C8, Acclaim PolarAdvantage (PA), and Acclaim PolarAdvantage II (PA). As can be seen from the wide range of polarity encompassed by these columns, they comprise a powerful column portfolio for routine application development. The polar characteristics of polarembedded phases often provide significantly better selectivity for mixtures of acids and bases. Generally speaking, you can expect acidic compounds to be retained longer and basic compounds to be retained slightly less on polarembedded phases than on conventional C8 phases. Figure shows a comparison of the Acclaim C8 column with the Acclaim PA column, run under identical conditions, illustrating this trend. The Acclaim PA column provides different and complementary selectivity to conventional C8 columns. Figure. Unique selectivity. Columns: µm,. mm Mobile Phase:. mm methanesulfonic acid Temperature: C Flow Rate:. ml/min Figure. Dewetting test in % aqueous eluent Conventional C8 Before stop-flow test After stop-flow test 8 Acclaim C8 Acclaim PA Column: See chromatograms, µm Dimensions:. mm Mobile Phase: CH CN/ mm potassium phosphate buffer, ph. v/v / Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks:. Uracil. µg/ml. Pyridine. Phenol. N,N-Dimethylaniline. Toluene. p-butylbenzoic acid Detection: UV, nm Peaks:. Cytosine. Uracil. Thymine Acclaim PA Before stop-flow test 8 Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: MeOH/ mm potassium phosphate buffer, ph. v/v 8/ Temperature: C Flow Rate:. ml/min Detection: UV, nm Sample: NIST SRM8 (column performance test mix for liquid chromatography) Peaks: Concentration Asymmetry (µg/ml) (EP). Uracil... Toluene.. Ethylbenzene 8.. Amitriptyline.. Quinizarin.. After stop-flow test Figure. Excellent peak shape for basic compounds.

4 Compatibility with % Aqueous Mobile Phases Chromatographers have learned, often the hard way, that conventional reversed-phase columns are not compatible with % aqueous mobile phases. For most conventional C8 columns, 9% aqueous is the limit before the phase collapses and retention is lost. Unfortunately, for many highly polar, water-soluble organic compounds, retention on a reversed-phase column requires the use of little or no organic modifier. The carefully-designed bonding technology of the Acclaim PA column incorporates hydrophilic functional groups between the hydrophobic alkyl chain and the silica surface that allows the surface to remain wetted, even in % aqueous mobile phase conditions. As shown in Figure, the conventional C8 column rapidly loses its ability to retain, following exposure to a pure aqueous buffer. In comparison, the Acclaim PA column is highly compatible with this aqueous environment, providing consistent and reliable retention times for even very hydrophilic nucleic acid bases such as cytosine, uracil, and thymine. Good Chromatographic Performance for Both Acidic and Basic Compounds At ph, amitriptyline is a sensitive probe for measuring silanol activity. At this ph, free silanols are negatively charged and the amitriptyline molecule is protonated (pk a = 9.). The protonated base interacts with ionized silanols by means of an ionexchange mechanism; the degree of tailing in this case is a direct measure of silanol activity. NIST 8 specifies both the chromatographic conditions and the nature of the test mixture for evaluating silanol activity on reversed phase silica columns. As shown in Figure, the Acclaim PA column provides a highly symmetrical peak for amitriptyline under this well-accepted testing protocol. Regardless, for Figure. Excellent peak shape for both basic and acidic compounds. Figure 8. High column efficiency. Acclaim PA XTerra RP8 Acclaim PA As.. Zorbaz Bonus-RP As.. Columns: See chromatograms, µm Dimensions:. mm Mobile Phase: CH CN Temperature: C Efficiency - plates/column Asymmetry -.8 Efficiency -8 plates/column Asymmetry -.9 Columns: See chromatograms, µm Dimensions:. mm Mobile Phase: CH CN/ mm potassium phosphate buffer, ph. v/v / Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks:. Uracil. µg/ml. Pyridine. Phenol. N,N-Dimethylaniline. Toluene. p-butylbenzoic acid Flow Rate:. ml/min Inj. Volume.: µl Detection: UV, nm Peaks: Efficiency - plates/column Efficiency -988 plates/column Asymmetry -. Efficiency - 9 plates/column Asymmetry -.8 Asymmetry -.. Uracil. Dimethylphthalate. Phenanthrene Efficiency - plates/column Asymmetry Batch A Batch B Batch C Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: CH CN/D.I. H O, v/v, / Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks:. Uracil. Dimethylphthalate. Phenanthrene Figure 9. Batch-to-batch reproducibility.

5 particularly difficult samples, comprising highly basic compounds and requiring a highly aqueous mobile phase, acidic conditions will produce the best results on the Acclaim PA column. Figure shows that Acclaim PA column provides excellent peak shapes for acidic, basic, and neutral compounds within a single run in a phosphate buffer at ph.. In comparison, the ZORBAX Bonus-RP column, featured as a hydrolytically stable polar-embedded phase, shows poor peak shape for p- butylbenzoic acid, despite the fact that neutral and basic compounds are eluted with symmetrical peak shapes.. Figure. Nucleic acid bases. 8 Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: mm potassium phosphate buffer, ph. Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks:. Cytosine µg/ml. Uracil. Adenine. Guanine. Thymine High Column Efficiency The combination of advanced bonding technology and optimal packing procedures results in superior column efficiencies for Acclaim PA columns. As illustrated in Figure 8, the Acclaim PA column provides higher efficiencies than other reversed-phase columns designed for high-ph applications, including the XTerra C8 column (Waters).. Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: CH CN/. M NH OAc, ph., v/v, / Temperature: C Flow Rate:. ml/min Inj. Volume: µl Detection: UV, nm 8 9 Peaks: ( ppm each). Sulfanilamide. Sulfathiazole. Sulfamerazine. Sulfamethazine. Sulfisoxazole. Sulfapyridazine. Sulfamethoxazole 8. Sulfadimethoxine 9. Sulfaquinoxaline sodium Good Batch-to-Batch Reproducibility Each Acclaim PA column is manufactured to stringent specifications to ensure column-to-column reproducibility (Figure 9). Each column is shipped with a lot validation sheet showing the test results and specifications for the lot of bonded silica packed into the column and an individual test chromatogram validating performance. All columns are individually tested for capacity and efficiency, and closely monitored for metal contamination. Figure. Sulfa drugs. Column: Acclaim PA, µm Dimensions:. mm. Mobile Phase: (A) CH CN (B) mm phosphate buffer, ph. Gradient: Hold B for min B to A/B (/) in min then to A/B (/) in min Hold A/B (/) for min Temperature: C 8 Flow Rate:. ml/min Inj. Volume: µl Detection: UV, nm Peaks:. Thiamine. µg/ml. Ascorbic acid 9. Pyridoxine.. Niacinamide. Pantothenic acid. Vitamin B.. Folic acid 8. Riboflavin 8 9. Biotin. 8 Figure. Water-soluble vitamins.

6 Broad Range of Applications The Acclaim PA columns can be used for a wide range of applications, as illustrated in Figures. Figures,, and show separations for nucleic acid bases, sulfa drugs, and water-soluble vitamins, respectively, in highly aqueous conditions with excellent resolution and peak efficiencies. Separations of fat-soluble vitamins and parabens (Figures and ) represent more conventional reversed-phase applications that are readily achieved on the Acclaim PA. Figures and demonstrate the potential of separating basic drugs, such as antidepressant drugs and beta blockers, at elevated ph to achieve optimum retention and peak shapes..8 Figure. Fat-soluble vitamins.. Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: CH CN/.% MSA, v/v, 9/ Temperature: C Flow Rate:. ml/min Inj. Volume: µl Detection: UV, nm Peaks: ( ppm each). Vitamin A acetate. Vitamin E acetate. Vitamin D. Vitamin D. Vitamin K. Vitamin E Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: CH CN/H O v/v / Temperature: C Flow Rate:. ml/min Inj. Volume: µl Detection: UV, nm Peaks: (. mg/ml each). Methylparaben. Ethylparaben. Propylparaben. Butylparaben 8 Figure. Parabens.. Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: MeOH/ mm triethylamine, ph.8 v/v / Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks: Concentration Asymmetry (µg/ml) (EP). Uracil.8. Doxepin.. Imipramine 8.. Amitriptyline. Figure. Antidepressants.

7 The versatility of the Acclaim PA column is clearly illustrated in the next series of chromatograms. Acidic mobile phases are used in Figures and 8 for the separation of peptides and cough syrup ingredients, respectively. Due to the nature of the cough syrup ingredients, highly aqueous conditions are necessary. In Figure 9, the Acclaim PA column is used for ion-pair chromatography for the baseline separation of nine catecholamines and their metabolites commonly found in urine. In Figure, the Acclaim PA column is used in an entirely different approach to separate anionic surfactants using a borate buffer and suppressed conductivity detection..8 Figure. Beta blockers. 8 Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: MeOH/.% NH OH, ph, v/v, / Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks: ( ppm each). Maleate. Labetalol. Metaraminol. Atenolol. Acebutolol. Metoprolol. Timolol 8. Oxprenolol. Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: Acetonitrile/.% methanesulfonic acid from /99 to / (v/v) in min Temperature: C Flow Rate:. ml/min Detection: UV, nm Peaks:. Gly-Tyr. Val-Tyr-Val. Met enkephalin. Leu enkephalin. Angiotensin II Figure. Peptide mix..8 Column: Acclaim PA, µm Dimensions:. mm Mobile Phase: Acetonitrile/ mm phosphate buffer, ph.9 from /9 to / (v/v) in min Temperature: C Flow Rate:. ml/min Inj. Volume: µl Detection: UV, nm Peaks:. Doxylamine. Pseudo-ephedrine. Acetaminophen. Dextromethorphan Figure 8. Active ingredients in cough syrup.

8 SPECIFICATIONS Phase: Polar-embedded Particle Size: and µm Pore Size: Å Surface Area: m /g Carbon load: % ph Range:. ORDERING INFORMATION To order in the U.S., call (8) -9 or contact the Dionex Regional Office nearest you. Outside the U.S., order through your local Dionex office or distributor. Refer to the following part numbers. Acclaim PA, µm analytical (. mm)... 8 (. mm)...89 (. mm)...9 Acclaim PA, µm analytical (. mm)...9 (. mm)...99 Acclaim PA, µm Guard Cartridges ( mm), each...9 (. mm), each..9 Acclaim Guard Kit (Holder and Coupler)...9 Acclaim SST Guard Cartridge Holder...9 Guard to Analytical Column Coupler...9 Column: Acclaim PA, µm Dimensions:. mm. Mobile Phase: Buffer (.98 g citric acid,. g NaOAc,. mg EDTA, ml of. M methanesulfonic acid)/ MeOH v/v 9/ Temperature: C Flow Rate:. ml/min Inj. Volume: µl Detection: dc amperometry [GC electrode, 8 mv] Peaks: (mg/ml each) na. VMA. HMPG 8 9. NE. E. DHBA. NMN. MN 8. DA 9. HV.. HIAA Figure 9. Catecholamines in urine. µs Figure. POE() Ammonium lauryl sulfate. Column: Acclaim PA, µm Dimension:. mm Mobile phase: Acetonitrile/ mm borate buffer v/v / Temperature: C Flow Rate: ml/min Inj. Volume: µl ( ppm) Detection: Suppressed conductivity detection (AMMS III) Sample: POE() Ammonium lauryl sulfate Discovery is a registered trademark of Supelco. SymmetryShield is a trademark, and XTerra is a registered trademark of Waters Corporation. Luna is a registered trademark of Phenomenex Inc. Acclaim and AMMS are registered trademarks of Dionex Corporation. Dionex Corporation Dionex Corporation Dionex U.S. Regional Offices Dionex International Subsidiaries 8 Titan Way Salt Lake City Technical Center Sunnyvale, CA (8) -8 Australia () 9 Austria () Belgium () - 9 Canada (9) 8-9 China (8) 8 8 P.O. Box West South, Suite A Westmont, IL () 89- Denmark 9 9 France 9 Germany -99- Italy () Japan () 88- Sunnyvale, CA Salt Lake City, UT Houston, TX (8) 8- Korea 8 8 The Netherlands () Switzerland () 99 United Kingdom () Atlanta, GA () -8 * Designed, developed, and manufactured under an NSAI registered ISO 9 Quality System. (8) - (8) 9-99 Marlton, NJ (8) 9-9 LPN 8 M / Dionex Corporation