Application Note 5 Determination of Water- and Fat- Vitamins in Nutritional Supplements by HPLC with UV Detection Introduction Vitamins are a well-known a group of compounds that are essential for human health. These compounds can be classified in two main groups water- and fat-soluble. Water-soluble vitamins include B group vitamins (V B V B V B V B5 and V B ) and ascorbic acid ( ) while the fat-soluble vitamins primarily include retinol (V A ) tocopherol (V E ) radiostol (V D ) and antihemorrhagic vitamin (V K ). These vitamins play different specific and vital functions in metabolism and can cause health problems when they are either lacking or in excess. The supply of vitamins for a healthy life depends on diet; however even foods that contain the necessary vitamins can have reduced vitamin content after storage processing or cooking. Therefore many people take a multi-vitamin tablet to supplement their diet. To ensure that these tablets contain the labeled amounts of vitamins there must be a quality control assay for these tablets. Reversed-phase HPLC is a technique well suited for vitamin analysis. Water-soluble vitamins are often determined using an aqueous mobile phase while the fat-soluble vitamins use organic-solvent mobile phases based on their very different solubility properties. Although simultaneous separation of water- and fatsoluble vitamins has been reported the differences in sample preparation and separation mode result in longer separation time for the detection of fat-soluble vitamins. Therefore the simultaneous separation method is inefficient for many samples. In this application note (AN 5) integrated and efficient methods for determining water- and fat-soluble vitamins in nutritional supplements were developed. Efficient and simple methods for the extraction and determination of the water-and fat-soluble vitamins were established and the separations were performed on the UltiMate HPLC system with Acclaim columns. Most regulated methods 4 and commonly reported HPLC methods 5- use different mobile phases and columns for water- and fat-soluble vitamins. The water-soluble vitamins including seven B group vitamins and were well-resolved on the Acclaim Polar Advantage (PA) II column using a phosphate buffer in the mobile phase. The silica-based column contains a polar-embedded stationary phase 9 on which the highly polar can be well-retained without using an ion-pairing reagent in the mobile phase. 4 The fat-soluble vitamins including V A V A acetate V E acetate V D V D V K and β-carotene were well-resolved on the Acclaim C column using an organic-solvent mobile phase. Method performance including linearity detection limits reproducibility and analyte recovery was evaluated for both methods. Five nutritional supplement samples were analyzed and the results were equivalent to the labeled values.
Equipment Dionex UltiMate HPLC system consisting of: DGP A Pump with SRD solvent rack with degasser WPS TSL Autosampler TCC- Thermostatted column compartment VWD-4RS UV-vis Detector Chromeleon Chromatography Data System. SR Kudos SKLH Ultrasonic generator Kudos Ultrasonic Instrumental Co. Shanghai China IKA MS Minishaker IKA Works Guangzhou China Thermo Orion 4A+ ph Meter Reagents and Standards Deionized water from Milli-Q Gradient A Acetonitrile ( CN) methanol ( OH) methyl tert-butyl ether (MTBE) and dichloromethane (CH Cl ) HPLC grade Fisher Potassium dihydrogen phosphate (KH PO 4 ) phosphoric acid (H PO 4 ) and potassium bicarbonate (KHCO ) analytical grade SCRC China Folic acid ( ) ascorbic acid ( ) phylloquinone (VK) tocopherol acetate (V E acetate) β-carotene retinol (V A ) and retinol acetate (V A acetate) 9% Sigma-Aldrich Thiamine (V B ) riboflavin (V B ) nicotinamide (V B ) pantothenic acid (V B5 ) pyridoxine ( ) cyanocobalamin (V B ) ergocalciferol (V D ) and cholecalciferol (V D ) 9% National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) China. Samples Five vitamin supplement samples (Brands to 5) were analyzed. The ingredients are listed in Table. Brands and were from two different pharmaceutical companies and Brands 4 and 5 were from a third company that produces special vitamin tablets for women children and the elderly. STANDARD PREPARATION Water- Vitamin Standards Water-soluble vitamin standards of V B V B V B5 V B and are prepared by accurately weighing to mg of the vitamin powder and adding to g of DI water to make stock solutions of. mg/ml for each vitamin. Because of the limited stability of it should be freshly prepared. Because of the limited solubility in water of V B and folic acid ( ) the concentration of stock solution of V B is decreased to.5 mg/ml in DI water; while that of is prepared using mm of KHCO instead of DI water to make a solution of.5 mg/ml. Fat- Vitamin Standards Fat-soluble vitamin standards of V A acetate V D V D and V E acetate were prepared by accurately weighing to mg of each and adding OH to to g to form stock solutions of. mg/ml for each vitamin. The standards for V K and β-carotene were prepared using acetone and CH Cl instead of OH. Because of the limited stability of β-carotene a stock solution of. mg/ml was freshly prepared every days. The well-prepared stock standards were stored at 4 C when not in use and the stock standards of fatsoluble vitamins were stored in the dark. Water-soluble vitamin working standards were prepared from the stock standards on the day of use by dilution with DI water. A mixture of OH-CH Cl (: v/v) was used for fat-soluble vitamins. Table. Ingredients in Nutritional Supplement Samples Brands to 5 Brand Brand (B-complex) Brand (for women) Brand 4 (for children) Brand 5 (for the elderly) Ca V B V B V B V B V B P V B V B V B V B V B K V B V B Cl V B5 V B5 Mg Fe V B V B V A acetate V A acetate V A acetate Cu V E * V E * Zn V D V D Mn V A acetate β-carotene I V D Ca Ca Ca Cr Biotin Fe Fe Fe Mo V E * Zn Zn Zn Se V K Sn Sn Sn Ni β-carotene Sn Si V Note: * V E acetate was detected in these samples but it is labeled as V E. Determination of Water- and Fat- Vitamins in Nutritional Supplements by HPLC with UV Detection
SAMPLE PREPARATION Extraction of Water- Vitamins Grind the tablets with a mortar and pestle. Place. g of accurately weighed ground powder for Brands and and. g of accurately weighed ground powder for Brands to 5 respectively into ml volumetric flasks; then add ml of water to each flask. After 5 min of ultrasonic extraction add water to the mark. Extraction of Fat- Vitamins Place.5 g of accurately weighed ground powder of Brands 4 and 5 into ml volumetric flasks respectively and add ml of OH-CH Cl (: v/v) to each flask. After 5 min of ultrasonic extraction add OH -CH Cl (: v/v) to the mark. The well-prepared sample solutions should be stored in the dark and diluted if necessary. Prior to injection filter the solutions through a. µm filter (Millex -GN). CONDITIONS For Determination of Water- Vitamins Column: Acclaim PA 5 μm Å 4. 5 mm (P/N 9) Temperature: 5 C Mobile Phase: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) In gradient (Table ) Flow Rate:. ml/min Injection Vol: μl UV Detection: Absorbance at 45 5 and nm (Table ) For Determination of Fat- Vitamins Column: Acclaim C 5 μm Å 4. 5 mm (P/N 59) Temperature: 5 C Mobile Phase: A: OH- CN (: v/v) B: MTBE In gradient (Table ) Flow Rate:. ml/min Injection Vol: μl UV Detection: Wavelength-switching absorbance at 5 5 and 45 nm (Table ) Table. Gradients for Water- and Fat- Vitamin Separations Water- Vitamins Fat- Vitamins Time (min) A (%) CN B (%) 5 mm Phosphate buffer (ph.) Time (min) A (%) OH- CN (:v/v) B (%) MTBE.. 95 5 4.. 95 5 4. 5 5 4.5 4.5 5. 5 5 9.. 5 5 9.5. 95 5. 5 95 5 Table. Detection Wavelengths for Water- and Fat- Vitamins Water- Vitamins Fat- Vitamins Detection Wavelength (nm) Analytes Switching Time (min) Detection Wavelength (nm) Analytes V B V B5 V B. 5 V A V A acetate 45 V B V B * 4. 5 V D V D V K V E acetate 5 V B 9. 45 β-carotene * Note. * and V B are detected at and 45 nm respectively when analyzing Brand (details shown in Sample Anaysis Water- Vitamins). RESULTS AND DISCUSSION Separation and Detection Water- Vitamins Some smaller acids such as formic and acetic are commonly used as mobile phase buffers and absorb at nm. For this reason the baseline absorbance shifts during the gradient as the amount of these acids in the mobile phase changes. Additionally initial experiments demonstrated that the retention of V B is inadequate when using formic acid unless an ion-pairing reagent is added to the mobile phase. Therefore a phosphate buffer was used to avoid the baseline shift and to retainv B. Application Note 5
The buffer s ph value may significantly affect the retention of water-soluble vitamins on the polar group embedded Acclaim PA column which is suitable for separation of compounds with high polarity. This is especially important for the separations of V B V B and. As shown in Figure the eight water-soluble vitamins are well separated at ph.. The water-soluble vitamins are a structurally diverse group of compounds with different absorbance maxima. Therefore as shown in Table four detection wavelengths were chosen for their detection. Fat- Vitamins Non-aqueous reversed-phase (NARP) is usually used for determining fat-soluble vitamins by HPLC so that the vitamins are soluble throughout the analysis. A typical NARP mobile phase consists of a polar solvent (usually acetonitrile) a solvent with lower polarity (e.g. dichloromethane) to act as a solubilizer and to control retention by adjusting the solvent strength and an amount of a third solvent with hydrogen-bonding capacity (e.g. methanol) to optimize selectivity. Therefore an acetonitrile methanol and MTBE mobile phase was used to separate fat-soluble vitamins using the Acclaim C column. Because these are all low-polarity compounds a C stationary phase is a good choice for their separation. Figure shows good resolution of seven fat-soluble vitamins: V A V A acetate V D V D V E acetate V K and β-carotene. The fat-soluble vitamins also are a structurally diverse group of compounds with different absorbance maxima. Therefore as shown in Table three detection wavelengths were chosen to determine them and wavelength-switching was employed in order to obtain a single chromatogram and increase detection sensitivity. 4 Column: Acclaim PA 5 µm 4. 5 mm Eluents: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) Gradient: 4 4 min 5% A 4.5 9 min % A 9.5 min % A Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: UV at nm Peaks: -V B - (Separated alone under the same chromatographic conditions) - 4-V B 5-V B5 -V B - -V B 4 5-4 4 Figure. Overlay of chromatograms of and V B group vitamins. 4 44 Column: Acclaim C 5 µm 4. 5 mm Eluents: A: OH- CN (: v/v) B: Methyl tert-butyl ether (MTBE) Gradient: 4.5 min 95 % A 5 min 5% A. 5 min 95% A Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: Wavelength-switching absorbance at 5 5 and 45 nm Wavelength- Switching Time:. min 5 nm 4. min 5 nm 9. min 45 nm Peaks: -V A -V A acetate -V D 4-V D 5-V E acetate -V K -β-carotene 5-4 4 5 45 Figure. Chromatogram of fat-soluble vitamins and β-carotene. 4 Determination of Water- and Fat- Vitamins in Nutritional Supplements by HPLC with UV Detection
Reproducibility Linearity and Detection Limits Prior to sample analysis the reproducibility was estimated by making six replicate injections for watersoluble vitamins and nine replicate injections for fatsoluble vitamins. Excellent RSDs for retention time and peak area are shown in Table 4. Calibration linearity for the water- and fat-soluble vitamins was investigated respectively by making three replicate injections of a mixed standard prepared at five or six different concentrations. The external standard method was used to calculate the calibration curve and to quantify these compounds in samples. Table 5 reports the data from the calibration as calculated by the Chromeleon software. We found linear calibration curves for each vitamin over the ranges that were evaluated. The single-sided Student s t test method was used for estimating method detection limits (MDL). These data are also reported in Table 5. Table 4. Reproducibility of Time and Peak Area for Water*- and Fat**- Vitamins Water- Vitamin Time RSD Peak Area RSD Fat- Vitamin Time RSD Peak Area RSD V B..4 V A..9 V B.. V A acetate.9.59 V B..59 V D.5.44 V B5..4 V D.5.9..5 V E acetate.9.49..5 V K..5 V B.4. β-carotene.4.59.. Note. * Seven consecutive injections for water-soluble vitamins. ** Nine consecutive injections for fat-soluble vitamins. Table 5. Calibration Data as Reported by Chromeleon Software and MDLs for the Water- and Fat- Vitamins Water- Vitamin Detection Wavelength (nm) Regression Equation r (%) RSD Range (µg/ml) MDL* (µg/ml) V B 45 A =. c -.5 99.99.. 5. V B 5 A =.9 c +. 99.999.49. 5. V B A =.5 c -.9 99.99.9. 5.4 V B5 A =. c -.5 99.5 5.4. 5.9 A =.55 c -.9 99.9.9.. 5 A =.55 c -.4 99.99.. A =. c -. 99.9..5 5. A =.9 c -. 99.9 4.44. V B. 5 45 A =.45 c -. 99.94.94. 45 A =.4 c -.599 99.99.4.. Fat-soluble Regression r (%) RSD Range MDL* (µg/ml) Vitamin Equation (µg/ml) V A acetate A =.49 c -.4 99.99..5 4. V D A =.995 c +. 99.99..5 4. V D A =.5 c +.4 99.99..5 4. V E acetate A =. c +. 99.99.4. 5 4.4 V K A =.445 c +. 99.99.9.5. β-carotene A = 5.5 c -.4 99.9 4.4.5 4. Note. * The single-sided Student s t test method (at the 99% confidence limit) was used for determining MDL where the standard deviation (SD) of the peak area of seven injections is multiplied by.5 to yield the MDL. Application Note 5 5
Water- Fat- Water- Fat- Analyte Table. Analysis Results of Water- and Fat- Vitamins in Nutritional Supplement Samples* Brand Brand Added Found Recovery (%) Added Found V B..94..9 9 5.59 5.4..4 9 V B...4. 9 5.59 5.49.. V B...4.9 9 4. 4...4 9 V B5..5.. 4. 4... 95...5. 99 5.59 5.5.. 95.... 99 / / / / / V B.4..4.4 5.5.5.5.5 4. 4..5 9 / / / / / V A acetate.. 5. 4. 4 V D... V D. V D / / / / V E acetate. 4. 4.. 9 V K...4.4 β-carotene...5.4 9 Brand Brand 4 Brand 5 Analyte Added Found Recovery (%) V B.5.49..95 95.5.49.5.4 V B.5.4..94 94.5.5.5.4 V B / / / / / / / / / V B5 / / / / / / / / /.5.4...5.4.5.49 Recovery (%)....9 95.5.5.5. V B / / / / / / / / / 5. 4.5 5. 4. 95 5. 4.5 5. 49.9 V A acetate.5.4.. no amount **.45 V D V D / /.5 / /.5 V D / /.. V E acetate 5. 5. /..5.9 β-carotene.5.4 /.5 /. Note. * Three injections for each (n = ) ** V A acetate is labeled in Brand 5 but its amount is not declared. Sample Analysis Water- Vitamins Water-soluble vitamins can be extracted from the nutritional supplement samples without causing chemical change by using water with ultrasonic extraction. Although (folic acid) and V B have limited solubility in water they still can be extracted effectively because of their low-levels in the samples. Good recoveries of watersoluble vitamins in the spiked sample (Table ) verified that water extraction is suitable; however some fatsoluble vitamins such as V E acetate V A and β-carotene can be partially extracted and their retention is very strong under the chromatographic conditions used for water-soluble vitamins analysis. Therefore after sample analysis the column should be washed using a mobile phase with a high-percentage of organic solvent. The frequency of this cleaning will vary with the sample the sample injection volume and the number of injections. Determination of Water- and Fat- Vitamins in Nutritional Supplements by HPLC with UV Detection
9 b A 4 Column: Acclaim PA 5 µm 4. 5 mm Eluents: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) Gradient: 4 4 min 5% A 4.5 9 min % A 9.5 min % A Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: UV at nm Samples: a: Sample Brand b: Spiked sample Brand Peaks: -V B - - 4-V B 5-V B5 -V B - -V B 5 Column: Eluents: Gradient: 5 Acclaim PA 5 µm 4. 5 mm A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) 4 4 min 5% A 4.5 9 min % A 9.5 min % A B b 4 5 Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: UV at 45 nm Samples: a: Sample Brand b: Spiked sample Brand Peaks: -V B - - 4-V B 5-V B5 -V B - -V B b a.5 - a a - 5 5 4-5 5 4 Column: Acclaim PA 5 µm 4. 5 mm Eluents: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) Gradient: 4 4 min 5% A 4.5 9 min % A 9.5 min % A 5 C 4 Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: UV at 5 nm Samples: a: Sample Brand b: Spiked sample Brand Peaks: -V B - - 4-V B 5-V B5 -V B - - V B Column: Acclaim PA 5 µm 4. 5 mm Eluents: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) Gradient: 4 4 min ~5% A 4.5 9 min % A 9.5 min % A 5 D Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl Detection: UV at nm Samples: a: Sample Brand b: Spiked sample Brand Peaks: -V B - - 4-V B 5-V B5 -V B - -V B 4 b 5 5 a - 5 5 4-5 5 4 Figure. Chromatograms of water-soluble vitamins in Brand collected at A) nm B) 45 nm C) 5 nm and D) nm. Five nutritional supplement samples were analyzed. Figure shows chromatograms of Brand which were collected at different detection wavelengths and the same sample spiked with standards. For Brand impurities may interfere with the detection of (peak ) and V B (peak ) at nm. Although these samples have Application Note 5
Column: Acclaim C 5 µm 4. 5 mm Eluents: A: OH- CN (: v/v) B: Methyl tert-butyl ether (MTBE) Gradient: 4.5 min 95 % A 5 min 5% A. 5 min 95% A Temperature: 5 C Flow Rate:. ml/min Injection Vol.: µl 5 5 Detection: Wavelength-switching absorbance at 5 5 and 45 nm Wavelength- Switching Time:. min 5 nm 4. min 5 nm 9. min 45 nm Samples: a: Sample Brand b: Spiked sample Brand Peaks: -V A -V A acetate -V D 4-V D 5-V E acetate -V K -β-carotene Samples: A: Water-soluble vitamins standards B: Fat-soluble vitamin standards Injection Vol.: 5 µl Temperature: 5 C For water-soluble vitamins: Column: Acclaim PA µm. 5 mm Eluents: A: CN B: 5 mm Phosphate buffer (dissolve about.4 g KH PO 4 in L water and adjust ph to. with H PO 4 ) Gradient:.5 4.5 min 45% A 4.5 5 min 45 % A 5 min % A Flow Rate:.9 ml/min Detection: Wavelength-switching absorbance at:. min 45 nm;. min nm;.4 min nm. min nm Peaks: -V B - - 4-V B 5-V B5 -V B - -V B b a 4 For fat-soluble vitamins: Column: Acclaim C µm 4. 5 mm Eluents: A: OH- CN (: v/v) B: Methyl tert-butyl ether (MTBE) Gradient:.5. min 95 % A..5 min 5% A 4.5 4. min 5 95% A Flow Rate:. ml/min Detection: Wavelength-switching absorbance at:. min 5 nm;. min 5 nm; 4.5 min 45 nm Peaks: -V A -V A acetate -V D 4-V D 5-V E acetate -V K -β-carotene - 4 4 4 A 4 Figure 4. Chromatograms of fat-soluble vitamins in Brand. 5 greater absorption at nm they are best detected at and 45 nm respectively in order to eliminate the interference. In the other samples no impurities interfered with the detection of and V B at nm. Analysis results of the five samples (i.e. recovery of analytes added to the samples and amounts of the water-soluble vitamins in each sample) are summarized in Table. The recoveries of the water-soluble vitamins ranged from 94% to % and the detected amounts were in agreement with the labeled values suggesting that the extraction and determination are accurate. Fat- Vitamins The fat-soluble vitamins were extracted from the nutritional supplement samples without causing chemical change. This was achieved through use of a solvent system (a mixture of dichloromethane and methanol) that was capable of effectively penetrating the sample matrix and was used in conjunction with ultrasonic extraction. Four nutritional supplement samples (Brands 4 and 5) were analyzed. Figure 4 shows chromatograms of Brand and the same sample spiked with standards - 4 5 45 B -5 4 5 4 Figure 5. Fast separation of A) water- and B) fat-soluble vitamins on the UltiMate system using the Acclaim HPLC columns packed with resins with smaller particle diameter. 5 4 which were collected at three different detection wavelengths using wavelength-switching. Analysis results of the four samples are summarized in Table. The detected amounts of fat-soluble vitamins are in agreement with the labeled value except for the amount of V K in Brand which was determined as 5% of the labeled value. Although the amounts of β-carotene and V E are not labeled they were found in Brand 4. The same was true for β-carotene in Brands and 5. Determination of Water- and Fat- Vitamins in Nutritional Supplements by HPLC with UV Detection
CONCLUSION HPLC is an efficient method for determining the vitamin content of nutritional supplements. After the appropriate sample preparation water-soluble vitamins are determined in less than min using an Acclaim Polar Advantage II column starting with a % aqueous mobile phase with no ion-pairing reagent. Fat-soluble vitamins are determined in under 5 min using the Acclaim C column and a highly organic mobile phase. APPENDIX Faster separations of water- and fat-soluble vitamins may be completed on the UltiMate A system using the Acclaim HPLC columns packed with resins with smaller particle diameter ( μm. 5 mm PA [P/N 5] for water-soluble vitamins and C [P/N 9] for fat-soluble vitamins). As shown in Figure 5 good separations are completed within 4. min for water-soluble vitamins and within 5. min for fatsoluble vitamins. See Tables and for the gradient and detector parameters for faster separations. Table. Gradients for Faster Water- and Fat- Vitamin Separations Water- Vitamins Fat- Vitamins Time (min) A (%) CN B (%) 5 mm Phosphate buffer (ph.) Time (min) A (%) OH- CN (:v/v) B (%) MTBE.. 95 5.5.5 95 5 4.5 45 55. 5..5 5 5. 4.5 5 5 4. 95 5 Table. Wavelength Switching Time for Faster Water- and Fat- Vitamin Separations Water- Vitamins Fat- Vitamins Switching Time (min) Detection Wavelength Switching Time (min) Detection Wavelength. 45 5.. 5.4 4.5 45. Application Note 5 9
References. Nollet Leo M.L. Food Analysis by HPLC nd Edition Marcel Dekker Inc. New York and Basel.. Dionex Corporation. Application Note LPN 45 Determination of Water- and Fat- Vitamins in Functional Waters by HPLC with UV-PDA Detection Sunnyvale CA 9.. Determination of Thiamine Hydrochloride Pyridoxine Hydrochloride Niacin Niacinamide and Caffeine in Health Foods GB/T 59 9-. 4. Determination of Vitamin A D E Content in Milk Powder and Formula Foods for Infant and Young Children GB/T 54.9-99. 5. Moreno P.; Salvado V. Determination of Eight Water- and Fat- Vitamins in Multi-Vitamin Pharmaceutical Formulations by High-Performance Liquid Chromatography J. Chromatogr. A 5.. http://www.dionex.com/en-us/images/pdfcgram/4-45-vitamins-d-d.pdf. http://www.dionex.com/en-us/images/pdfcgram/5-55-fat-soluble-vitaminscarotenoids.pdf. http://www.dionex.com/en-us/images/pdfcgram/5-4-fat-soluble-vitaminstandards.pdf 9. Dionex Corporation. Acclaim Polar Advantage II (PA) HPLC Columns Data Sheet LPN Sunnyvale CA 5.. Wu S.J.; Zhuang Z.H.; Zhu M.L.; Xu X.Y. Determination of Six Water- Vitamins and Caffeine in Health Foods by HPLC Chin. J. Chromatogr. 4() 9. Acclaim Chromeleon and UltiMate are registered trademarks of Dionex Corporation. IKA is a registered trademark of IKA Works. Kudos is a registered trademark of Kudos Ultrasonic Instrumental Co. Millex and Milli-Q are registered trademarks of Millipore Corporation. Passion. Power. Productivity. Dionex Corporation North America Europe Asia Pacific Titan Way U.S./Canada (4) 95-5 Austria (4) 5 5 Benelux () 9 () 5 494 P.O. Box Denmark (45) 9 9 France () 9 Germany (49) 99 Sunnyvale CA Ireland (5) 44 4 Italy (9) 5 Sweden (4) 4 Taiwan () 5 55 South America 94- Switzerland (4) 5 99 United Kingdom (44) 9 Brazil (55) 54 (4) - www.dionex.com Australia () 94 5 China (5) 4 India (9) 4 5 Japan () 5 Korea () 5 5 Singapore (5) 9 9 LPN 5 PDF / Dionex Corporation