Acclaim C30 Column. Standards in Isopropanol. Saponified Chicken Fat Minutes

Transcription

1 columns C Column Standards in Isopropanol 4 Saponified Chicken Fat Peaks:. SDA. EPA. ALA 4. GLA. DHA. Arach. 7. LLA 8. DPA 9. 9E, 4Z-CLA. ETA. Adrenic. Oleic. EDA 4. Erucic Corona ultra detector 88 C columns provide unique selectivity and superior separations of hydrophobic, structurally related analytes. Columns for Separating Hydrophobic Structurally Related Isomers The C is designed to provide high shape selectivity for separating hydrophobic structural related isomers and unique selectivity complementary to other reversed-phase columns (e.g. C8). It is based on covalent modification of C alkyl silane on high-purity, spherical, porous silica gel. The combination of advanced surface modification technology and careful matching of C alkyl chain with the pore size of the silica substrate provides the following benefits: High shape selectivity Unique selectivity complementary to other reversed-phase columns Compatibility with highly aqueous mobile phases High-quality columns with low column bleed, high efficiency, and rugged packing Passion. Power. Productivity.

2 High Shape Selectivity The C column provides significantly greater shape selectivity compared to C8 columns, making it suited to separating hydrophobic structural isomers, such as carotenoids, tocopherol, etc. Shape selectivity can be characterized by elution order of three polycyclic aromatic hydrocarbons (PAHs) in acetonitrile: benzo[a] pyrene (BaP),,:,4:,:7,8-tetrabenzonaphthalene (TBN, alternate name, dibenzo[g,p]chrysene), and phenanthro[,4-c]phenanthrene (PhPh) described in NIST SRM 89a. A smaller α (TBN/BaP) (= kʹtbn / kʹbap ) value indicates higher shape selectivity. As shown in Figure, the C column exhibits the highest shape selectivity of all reversed-phase columns. Unique Selectivity Complementary to Other Reversed-Phase Columns Selectivity is the key for successful separation. While C8 columns are most commonly used for small molecule separations, other reversedphase columns with different selectivity are often needed for optimal result. Compared to other types of reversedphase columns including C8, polar-embedded, and phenyl phases the C demonstrates different selectivity and provides a complementary tool for method development (Figure ). % Aqueous Compatibility To separate hydrophilic analytes, such as organic acids and water-soluble vitamins on a reversed-phase column, a highly aqueous mobile phase is often required to achieve adequate retention. However, most C8 columns will dewet under such condition, resulting in unreliable results. Due to a rigid and highly ordered C alkyl chain, along with the use of a wider pore silica substrate, C columns exhibit % aqueous compatibility no loss of retention was observed after stopflow cycles (see Figure ). α (TBN/BaP) α (TBN/BaP) = / kʹtbn kʹbap C C8 PA Column Name PA Phenyl- Figure. Shape selectivity comparison. Note: lower (TBN/BaP) suggests higher shape selectivity. AU C Phenyl Polar Advantage Polar Advantage II C8, 9 Figure. The unique selectivity of the column family. Retnetion of Thymine (min) 8 4 De-wetting Test 4 Number of Stop-Flow Cycle Figure. Aqueous compatibility of the C column. Columns: (4. mm) Mobile Phase: Acetonitrile/water 8: v/v Temperature: C Flow Rate: ml/min Inj. Volume: µl Detection: UV (4 nm) Sample: NIST SRM 89a standard mix. Benzo[a]pyrene (BaP). Phenanthro[,4-c]phenanthrene (PhPh). Dibenzo[g,p]chrysene (TBN) Columns: (4. mm) Mobile Phase: Acetonitrile/water 8: v/v Temperature: C Flow Rate: ml/min Inj. Volume: µl Detection: UV (4 nm) Sample: NIST SRM 89a standard mix. Benzo[a]pyrene (BaP) 87. Phenanthro[,4-c]phenanthrene (PhPh). Dibenzo[g,p]chrysene (TBN) 87 Column: C, µm (4. mm) Mobile Phase: mm Ammonium acetate, ph Temperature: C Flow Rate: ml/min Inj. Volume: µl Detection: UV (4 nm) Sample:. mg/ml Probe: Thymine Stop-Flow Protocol:. Test the column under above condition. Stop the pump for min (pump pressure drops to ). Resume the flow and condition the column under above condition for min 4. Inject the sample and run the test under the same conditions.. Repeat through 4 87

3 High Quality of Column Manufacturing Low column bleed is essential for good detection limits, especially for mass spectrometer (MS) and charged aerosol detectors (CAD). The proprietary bonding process ensures the C bonded phase maintains good hydrolytic stability with extremely low column bleed. Our innovative packing method provides high column efficiency and robust column packing for most challenging applications. Each column is manufactured to strict specifications to ensure column-to-column reproducibility. In addition, each column is individually tested and shipped with a qualification assurance report. Wide Range of Applications The C column is a reversed-phase HPLC column. Like C8 columns, the C can be used in a broad range of applications including food and beverage, chemical, environmental, pharmaceutical, academia, etc. The also offers several unique features that set it apart from C8 columns. First, it exhibits higher shape selectivity suited to separation of hydrophobic, long-chain, structural isomers (e.g., carotenoids, steroids, etc). Second, it is fully compatible with various aqueous buffers, resulting in a broader application range (e.g., water-soluble vitamins, organic acids, etc) and more flexibility in method development. Third, it can be a viable alternative to normal-phase columns for lipid analysis. Therefore, the C be used as a generalpurpose column that complements C8 columns, and also serve as a specialty column when a C8 column fails to provide satisfactory results. Carotenoids Carotenoids are occur naturally in the chloroplasts and chromoplasts of plants, and some fungi and bacteria. They serve two key roles in plants and algae: ) they absorb light energy for use in photosynthesis, and ) they protect chlorophyll from photodamage. In humans, four carotenoids (beta-car- 8 mau C B A nm 8 Reference: HarvetsPlus Handbook for Carotenoid Analysis, D.B. Rodriguez-Amaya and M. Kimura, International Food Policy Research Institute, 4. Figure 4. Carotenoids in vegetables. Figure. Glucocorticosteroids β-carotene 4 otene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin) have vitamin A activity, and can also act as antioxidants. As shown in Figure 4, six common carotenoids and chlorophyll are separated on the C column with excellent selectivity and resolution. Glucocorticosteriods Glucocorticosteroids are a group of naturally occurring and synthetic hormones that moderate inflammation and other stress responses. These compounds 7 8 Column: C, µm (4. mm) LC System: UltiMate RS Mobile Phases: A. Acetonitrile B. Methanol/ethyl acetate : (v/v) C. mm Acetic acid in water Gradient: Time (min) %A %B %C Flow:. ml/min Temperature: C Inj. Volume: 8 µl Detector: Diode array; VIS 4 nm, spectra 8 nm Samples: A. Maize extract in acetone B. Spinach extract in acetone C. Carrot extract in acetone Peaks:. Lutein. Zeaxanthin. Chlorophyll-b 4. alpha-cryptoxanthin. beta-cryptoxanthin. Chlorophyll-a 7. alpha-carotene 8. beta-carotene 4 Column: C, µm (. mm) LC System: UltiMate RS Mobile Phase: Methanol/tetrahydrofuran/water ::7 (v/v) Flow:. ml/min 7 Temperature: C mau Inj. Volume: µl 8 Detector: Diode array; UV 4 nm Peaks:. Prednisone µg/ml. Cortisone. Prednisolone 4. Hydrocortisone. Dexamethasone. -Methylprednisolone 7. Corticosterone 8. -Deoxyhydrocortisone 4 8 Reference: McWhinney B C, Ward G, Hickman P E, Clin. Chem, 99, 4, are included on the World Anti-Doping Agency s list of substances prohibited in competition. Separation of these substances is challenging due to their structural similarity. The method of McWhinney is commonly used in clinical laboratories for monitoring of these substances. While the original method uses a C8 column, the C offers improved resolution and throughput with the same elution order used the literature method. Figure shows baseline separation of eight glucocorticosteroids using the C column.

4 Water-soluble Vitamins Water-soluble vitamins are essential nutrients. They are found naturally in foods, and they are also added to food, and formulated supplements. Chemically, they are a heterogeneous group of anions, cations, zwitterions, and neutrals. Due to the variety and complexity of matrices where vitamins are found, their analysis is often challenging. The C column demonstrates both high hydrophobic retention and aqueous compatibility; this desirable combination of features enables simple reversed-phase separation of these complex analytes (Figure ) Folic acid mau nm 4 7 Column: C, µm (4. mm) LC System: UltiMate RS Mobile Phases: A. Acetonitrile B. 7 mm Phosphoric acid + 4 mm sodium sulfate (ph.) Gradient: Time (min) %A %B Flow Rate:. ml/min Temperature: C Inj. Volume: µl Detection: Diode array; UV nm, spectra 4 nm Samples: Vitamin standards in mobile phase Peaks:. Thiamine µg/ml. Ascorbic acid. Niacinamide 4. Pyridoxine.Pantothenic acid. Folic acid. 7. Riboflavin. 87 Triglycerides in Cooking Oils Cooking oils are purified lipids from plants, and they are typically liquid at room temperature. These compounds contain triglycerides as major components, and small quantities of free fatty acids, mono-, and diglycerides. The composition of cooking oils is highly complex due to the wide variety of alkyl chain length, degree of unsaturation, origin, etc. While normalphase chromatography is often used to characterize oils by their hydrophilicity, reversed-phase chromatography provides high resolution for analyzing major and minor components, and obtaining a detailed fingerprint. Due to its high shape selectivity, the C provides higher resolution than the C8 column for oil analysis (Figure 7). Separations of several cooking oils are illustrated in Figure 8. Figure. Water-soluble vitamins. B. C8 A. C Figure 7. Analysis of cooking oil: C vs. C8. C B A Columns: A. C, µm (4. mm) B. C8, µm (4. mm) Mobile Phase: Acetonitrile (CH CN)/Iso-propanol (IPA)/ Ammonium acetate buffer (. M, ph.) Gradient: Time (min) CH Temperature: 4 C Flow Rate:. ml/min Inj. Volume: µl Detection: Corona ultra (gain = ; filter = medium; neb. temp. = C) Sample: Peanut oil ( mg/ml in iso-propanol) 877 Column: C, µm (4. mm) Mobile Phase: Acetonitrile (CH CN)/iso-propanol (IPA)/ ammonium acetate buffer (. M, ph.) Gradient: Time (min) CH Temperature: 4 C Flow Rate:. ml/min Inj. Volume: µl Detection: Corona ultra (gain = ; filter = medium; neb. temp. = C) Samples: A. Peanut oil B. Olive oil C. Wheat germ oil ( mg/ml in iso-propanol) 878 Figure 8. Analysis of cooking oils. 4

5 Phospholipids in Egg Lecithin This class of lipids are major components of all cell membranes as they can form lipid bilayers. Most phospholipids contain a diglyceride a phosphate group connected to a hydrophilic organic group such as choline. The first phospholipid identified in biological tissues was lecithin in the egg yolk. Analysis of phospholipids is challenging due to its complexity (hydrocarbon length, degree of saturation of diglyceride moiety, and type of the hydrophilic organic group). Figure 9 shows the profile of lecithin from egg yolk and soybean obtained using an C column with a Corona ultra CAD detector. It indicates that lecithin from egg yolk contains both phospholipids and triglycerides, with minor impurities. To obtain detailed information on phospholipid composition, a higher-resolution method can be used (Figure ). Omega Fatty Acids Omega- fatty acids are a family of unsaturated fatty acids that share a final carbon carbon double bond in the n- position. Omega- fatty acids, such as α-linolenic acid (8:, n-; ALA), eicosapentaenoic acid (:, n-; EPA), and docosahexaenoic acid (:, n-; DHA), are important in human nutrition. The biological effects of Omega- are largely mediated by their interactions with the Omega- fatty acids. Some medical research suggests that excessive levels of Omega- relative to Omega- fatty acids may increase the probability of a number of diseases and depression. As shown in Figure, the C column exhibits excellent resolution power for a suite of closely related Omega fatty acids. Degradants Phospholipids 4 48 Figure 9. Profile of egg lecithin. Degradants Phospholipids Triglycerides 9 Figure. Phospholipids in egg lecithin. A ng LLA Area B Column: C, µm (4. mm) Mobile Phase: Acetonitrile (CH CN)/iso-propanol (IPA)/ ammonium acetate buffer (. M, ph.) Gradient: Time (min) CH Temperature: 4 C Flow Rate:. ml/min Inj. Volume: µl Detection: Corona ultra (gain = ; filter = medium; neb. temp. = C) Sample: Egg lecithin PLS ( mg/ml in iso-propanol) 879 Column: C, µm (4. mm) Mobile Phase: A. Acetonitrile (CH CN) B. Iso-propanol (IPA) C. Ammonium acetate buffer (. M, ph.) Gradient: Time (min) CH Temperature: 4 C Flow Rate:. ml/min Inj. Volume: µl Detection: Corona ultra (gain = ; filter = medium; neb. temp. = C) Sample: Egg lecithin PLM ( mg/ml in iso-propanol) 88 Column: C, µm (4. mm) LC System: UltiMate RS, Dual Gradient Mobile Phases: A. Water:formic acid:mobile phase B 9:.: (v/v) B. Acetone:acetonitrile:THF:formic acid 7:::4(v/v) Gradient: Time (min) %A %B Flow Rate:. ml/min Temperature: C Inj. Volume: µl Detection: Corona ultra, nebulizer C, filter high Samples: A. Standards in isopropanol B. Saponified chicken fat Peaks:. SDA 8. DPA. EPA 9. 9E, 4Z-CLA. ALA. ETA 4. GLA. Adrenic. DHA. Oleic. Arach.. EDA 7. LLA 4. Erucic 88 Figure. Omega fatty acids.

6 Column Chemistry: C alkyl SPECIFICATIONS Silica Substrate: Spherical, high-purity Particle sizes: and µm Surface area: m /g Pore size: Å Operating ph range: ph 8 Operating Temperature: Up to C Aqueous compatibility: to % Solvent compatibility: to % Operating pressure and flow rate: See table top right Column Dimensions Operating Pressure and Flow Rate Specifications Particle Size Part Number Maximum Pressure (psi) ORDERING INFORMATION In the U.S., call (8) 4-9 or contact the Dionex Regional Office nearest you. Outside the U.S., order through your local Dionex office or distributor. Refer the following part numbers: C Analytical and Guard Columns Typical Flow Rate (Recommended) (ml/min) Maximum Flow Rate (ml/min) 4. mm µm 778 9, mm µm 779 8, mm µm 77 8,.8... mm µm 77,.4... mm µm 774 8,.4... mm µm 77 8,8... Part Number C, µm Analytical, 4. mm C, µm Analytical, 4. mm C, µm Analytical, 4. mm C, µm Analytical,. mm C, µm Analytical,. mm C, µm Analytical,. mm C, µm, Guard, 4. mm C, µm, Guard,. mm C, µm, Guard,. mm Accessories Part Number Guard Holder (V-) Guard Holder kit V- (Holder V- and Coupler) Guard Coupler Corona,, CAD, and UltiMate are registered trademarks, and ultra is a trademark of Dionex Corporation. Dionex Corporation North America Europe Asia Pacific 8 Titan Way P.O. Box U.S./Canada (847) 9-7 Austria (4) Benelux () () 494 Denmark (4) 9 9 France () 9 Germany (49) 99 Sunnyvale, CA South America Ireland () 44 4 Italy (9) 7 Sweden (4) Taiwan (88) Brazil () 7 4 Switzerland (4) 99 United Kingdom (44) 7 97 (48) Australia () 94 China (8) 48 8 India (9) 74 7 Japan (8) 88 Korea (8) 8 Singapore () 89 9 LPN 77 PDF / Dionex Corporation