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212_003 #01ALEXYS Iodide AnalyzerALEXYS Iodide AnalyzerERROR! REFERENCE SOURCE NOT FOUND.212_003 #01THE SOUNDEST LC-EC APPLICATIONS FORCLINICAL & DIAGNOSTICS ANALYSISPART NUMBERS AND CONFIGURATIONSEVER BUILD190.0035 ALEXYS 110 LC-EC system with DECADE II SDC115.4150 SenCell GC, salt bridge, 5/60RE.2000 ClinRep® complete kit , Catecholamine in urineClinRep® Analytical columnRE.2030Catecholaminesfor catecholamines in urineSerotoninRE.8021 ClinChek® urine control, level IMetanephrinesVMAHVA5-HIAA HomocysteineGlutathione(di-)sulfidesIodideVitamins A, C, D, E, and KQ10UbiquinolsINTRODUCTIONA method for the determinationof iodide is developedusing electrochemical detection(ECD) with a silver workingelectrode (WE). The methodconsists of ion exchangechromatography using aphosphate/citrate buffer at pH6.5, followed by amperometricdetection at 0.15 V.Detection limit of iodide is0.2-1 µmol/L (25 - 127 ng/mL)depending on the columnperformance. Reproducibilityof the determination of iodideis concentration depended. At50 µM RSD in peak area’s andheights was better than 2%.Below 1 µM the RSD valuesincreased to 10 – 22 %.Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comPage : 3/4• Iodide in Urine• Diagnosis of thyroid dysfunction• Excellent sensitivity and peak shape withMagic DiamondMethodThe HPLC system consists of a anion exchange column with aphosphate/citrate mobile phase (Table I). The use of halide ionssuch as chloride and bromide must be avoided as they are reactivetowards the silver electrode, causing a high backgroundcurrent and decreased sensitivity.Di-sodium phosphate and citric acid (both 10 mM) are dissolved in800 mL water, pH is set to 6.5 with NaOH and water is added to900 mL. Finally 10% methanol (100 mL) is added.Determination of analytes in complex matrices such as urine orplasma is often complicated by sample pre-treatment proceduresto improve the selectivity of a method. Without suitable pretreatmentco-eluting peaks make reliable and reproducible analysisimpossible.An exception is the determination of iodide in urine. Due to theselectivity of the silver working electrode, urine could be injecteddirectly. After dilution and filtration over a 0.2 µm membrane filter,urine samples have been injected onto the analytical column andanalysed.Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comPage : 1/4Page : 2/4nA2 . 52 . 01 . 51 . 00 . 5214_001 #01IS0 . 00 2 4 6 8 10 12 14 minFig. 1 Analysis of 20 μL ClinCal® urine calibrator. Concetration ofcatecholamines in the calibrator sample: 123 μg/L NA, 29.5 μg/LA and 227 μg/L Dopamine.The quantification of the catecholamines in the urine samples isperformed by means of a single-point calibration method using aurine calibrator. The ClinCal® urine calibrator supplied in theClinRep® kit is a lyophilised urine sample with a known amountof catecholamines. The urine calibrator should be processed viathe same sample preparation method as the urine samples. Anexample chromatogram of a urine calibrator analysis is shown infigure 1.An internal standard method is used to compensate for recoverylosses during the sample preparation step. To every urine sample,calibrator or control 30 μL of internal standard (IS) solution isadded. The IS response of the samples is compared to that of adirectly injected standard solution (ClinTest® standard) to determinethe recovery. The sample response is then interpolated to100% recovery to establish the real catecholamine concentrationin the urine samples.Table 1Set-upHPLCFlow cellColumnTable 2LC-EC conditionsFlow rateSampleCLINREP® KIT FOR THE ANALYSIS OF CATECHOLAMINESIN URINEALEXYS 110 LC-EC system with DECADE IISDC (p/n 190.0035)GC type flow cell with Ag/AgCl saltbridge REFClinRep® Analytical column for catecholaminesin urine1.0 mL/min20 µl, extracted with ClinRep® sample preparationcolumnsMobile phase ClinRep® catecholamine buffer#Temperature* TD2 SDC 30°C (separation & detection),E-cellTAS110 4°C (sample cooling)Range500 mV (vs. Ag/AgCl sat’d)I-cell10 nA/VADFCa. 0.2 – 3.0 nAAnalysis time 0.1 HzFlow rate15 minutes#) mobile phase was recycled during experiments. *) minimum actual oven & traytemperature which can be reached is dependent on ambient conditions.Analysis of ClinChek® controlsFor quality control of the analytical determination RecipeClinChek® urine controls have been used in both the normal(level I) and the pathological range (level II).nA1.81.61.41.21.00.80.60.40.20 2 4 6 8 10 12 14 minFig. 2. Overlay of 6 chromatograms of 20uL injections ofClinChek® control level I.Table I. Calculated concentration of urine controls level I and II(n=6). Concentration range specified by Recipe is given for reference(source: data sheet supplied with controls).Component Specified conc (µg/l) Calculated RSDMin Max conc (µg/l) (%)Control level INoradrenaline 44 66 58.7 0.8Adrenaline 14 21 18.7 0.8Dopamine 120 180 176.6 0.8Control level IINoradrenaline 125 187 156.9 0.2Adrenaline 29 43 35.1 1.5Dopamine 186 278 236.9 0.5The control samples are lyophilised urine samples which have tobe processed in the same way as the urine samples. Both ControlI and Control II were analysed and the analyte concentrationsquantified using the ClinCal urine calibrator. For both urine controlslevel I and II the determined concentrations were within theconcentration ranges specified by Recipe on the urine control datasheet (see table I).Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comPage : 2/4synthesised in the chromaffinCatecholaminescells of the adrenal medullaSerotoninand the sympathetic nervousToday, LC-EC has been established as a fast and reliable methodMetanephrinessystem and play an importantfor the determination of catecholamines and metabolites in plasmaand urine [1-5].VMAHVApart as neurotransmitters andThe ClinRep® complete kit for catecholamines in urine of Recipe5-HIAA Homocysteinein metabolic regulation. Tumorsof the sympathoadrenal preparation and routine analysis of urinary catecholamines. In thisGmbH (Munich, Germany) is a standardised kit for the sampleGlutathione(di-)sulfidessystem cause an elevation inapplication note the results are reported from analysing urineIodidesamples using a ClinRep® kit with an ALEXYS 110 LC-EC system.production and in urinaryVitamins A, C, D, E, and Kexcretion of catecholaminesQ10MethodUbiquinolsand their metabolites.The quantitative determina-One Recipe ClinRep® complete kit contains all the neces-sarytion of urinary catechola-chemicals and (calibration) materials for sample preparation andanalysis of 100 assays, excluding the analytical column. Urinemines is a rapid and precisesamples are processed using the following sample preparationdiagnostic method for theprocedure prior to analysis:identification of pheochromocytomaand other tumor dis-• 3 mL acidified urine sample (10 mL conc. 32% HCl per literurine) or urine calibrator is mixed with 10 mL stabilising reagentS and 30 μL internal standard (IS) and subsequently adeasesof the nervous systemsuch as neuroblastoma andjusted to a pH 3.0 – 7.0 using 0.5M NaOH.214_001 #01ganglioneuroma. Approx-• The mixture is applied to a ClinRep® sample preparation columnsto trap the catecholamines present in the sample.CLINREP® KIT FOR THE ANALYSIS OF CATECHOLAMINESimately, half of all pheochro-IN URINE• The column is subsequently washed with 15 mL HPLC-grademocytoma patients sufferwater to remove interfering compo-nents.from permanent hyperten-• 6 mL of eluting reagent E is then used to elute the catecholaminesfrom the extraction Page column. : 3/4sion, in rest episodic hypertensivecrises occurs. In• The eluate is collected, mixed and 20 μL injected in the LCabout 40 % of the latter groupsystem.plasma cate-cholamine concentrationsare not raised innoise. The method is linear for the determination of the catecholaminesAnalysis of urine samplesthe interval betweenin thetwoconcentration range from 1 – 1000 μg/L [6].Urine samples were collected from an apparently healthy volunteer.and analysed multiple times to determine the recoveries, mination To determine of catecholamine the inter-assay precision an urine sample (C) wascrises. Nevertheless, deter-LOD, intra- and inter-assay precision of the method.worked-up 4 times and analysed (duplicate injection), this procedurewas repeated the next day and the relative standard devia-levels in the 24-hours urineThe intra-assay precision of the method was determined using two allows tion the calculated. detection of pathologicallyincreased values,urine samples (A and B). The urine samples were worked-up 5times and duplicate analysis were performed to determine the Table III. Inter-assay precision (urine sample C,even after a hypertensiverelative standard deviation (RSD, %).n= 4 (samples) x 2 (duplicate injections) Catecholamines x 2 (days).crisis.Component RSD (%) Conc. (µg/l)Table II. Intra-assay precision of urine sample A and B,Sample Cn= 5 (samples) x 2 (duplicate injections).Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 Norepinephrine - 7502 Connelley Drive 3.8 - Hanover MD 21076 48.7 - USA - info.usa@myantec.com - www.myantec.comEpinephrine 6.2 5.1Component RSD (%) Conc. (µg/l)Dopamine 3.2 225.1Sample ANorepinephrine 2.8 30.6The RSD’s for norepinephrine and dopamine were smaller thenEpinephrine 1.7 16.04%. For epinephrine, which was present in the sample in a significantlylower concentration, the RSD was slightly higher,Dopamine 3.7 102.56.2%.Sample BNorepinephrine 2.5 20.7Epinephrine 14 3.6Dopamine 2.3 115.2nA1.41.21.00.80.60.40.20.0214_001 #01THE SOUNDEST LC-EC APPLICATIONS FORCLINICAL & DIAGNOSTICS ANALYSISEVER BUILD0.250.200.150.100.052 4 6 8 10 12 14 minnA2 3 4 5 6 7 minFig. 3. Overlay of 10 chromatograms of 20uL injections of urinesample B. Top-right: zoom in on NA and A peaks.The RSD’s for all components were typically smaller then 4%.Only for low concentrations of epinephrine, near the limit of quantitation,a RSD of 14 % was found.For all urine samples, controls and calibrator recoveries typicallyin the range of 85 – 95% were found, compared to a directlyinjected standard. The concentration limit of detection (CLOD) forthe method was approximately 1 μg/L for all catecholamines. TheCLOD here is based on a 20 µL injection and defined as the concentrationthat gives a signal that is three times the peak-to-peakINTRODUCTIONThe catecholamines norepinephrine(NA), epinephrine(A) and dopamine (DA) areCONCLUSIONThe ClinRep® complete kit for catecholamines in urine providesa standardised method for the sample preparation ofurine samples and fast & reliable analysis of urinary catecholaminesusing LC-EC.Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.com1• Important part as neurotransmitters• Tumors of the sympathoadrenal systemPage : 1/4ALEXYS AnalyzersAlexys clinical 110 & diagnosticsStandardised ALEXYS for Analyzers routine analysis with fluidly running applicationsALEXYS Catecholamines Analyzer• validated concept• robust, dedicated for routine analysis• tumor and cardiovascular markersToday, LC-ECD has been established as a fast andreliable method for the determination of catecholaminesand metabolites in plasma and urine as well forSerotonin. Complete kits contains all the necessarychemicals and (calibration) materials for samplepreparation and analysis of 250 plasma assays,excluding the analytical column.NorepinephrineEpinephrineDopamineNorepinephrineEpinephrineInternal StdDopamineNorepinephrineEpinephrineInternal StdDopamineNorepinephEpinephrineCLINICAL & DIAGNOSTICSAPPLICATION NOTECLINREP® KIT FOR THE ANALYSIS OFCATECHOLAMINESIN URINEALEXYS Iodide AnalyzerProven analyzers to guarantee your performance• Iodide in Urine• diagnosis of thyroid dysfunction• Excellent sensitivity and peak shape with Magic DiamondIODIDE IN URINECLINICAL & DIAGNOSTICSAPPLICATION NOTEWith the introduction of conductive diamond aninteresting new electrode material became availablein electrochemistry. Diamond has severaladvantages over conventional electrode materialssuch as a wide potential window in aqueoussolutions, excellent chemical inertness andstability. For this application a sensitive and reliableLC-ECD method is available for the analysis ofiodide based on DC amperometry using theMagic Diamond TM (MD) electrode.®10
220_002 #03ANALYSIS OF CARBOHYDRATESTHE FINEST LC-EC APPLICATIONS FORFOOD & BEVERAGE ANALYSISEVER PROCESSEDBisphenol ACatechinsPhenolsAntioxidantsPolyphenolsResveratrolEpicatechinQuercetinIodideQ10ubiquinolsPage : 2/10220_002 #03The retention behaviour of the carbohydrates can be controlled bythe concentration of sodium hydroxide and sodium acetate in themobile phase. An increase of the sodium hydroxide concentration[OH-] has a dual effect on the retention of carbohydrates. Theincrease in ionic strength of the eluent causes a decrease inanalyte retention, while the higher pH will increase the degree ofdissociation resulting in an increase in analyte retention. If the pH> pKa (full dissociation) then the ionic strength will dominate theseparation process and the retention decreases. This is illustratedin Fig. 1A.ABCABCX Axis Title0 5 10 15 20 25 30Minutes0 10 20 30MinutesANALYSIS OF CARBOHYDRATESANION-EXCHANGE CHROMATOGRAPHY AND PULSED AM-PEROMETRIC DETECTIONA. 50 mM NaOH - 1 mM NaOAcB. 30 mM NaOH - 1 mM NaOAcC. 15 mM NaOH - 1 mM NaOAcA 30 mM NaOH - 10 mM NaOAcB 30 mM NaOH - 2 mM NaOAcC 30 mM NaOH - 1 mM NaOAcFig. 1. [A] Retention times of common food carbohydrates as a function ofsodium hydroxide (top) and [B] sodium acetate (bottom) concentration ofthe mobile phase.Sodium acetate is commonly used to decrease the elution time ofhigher molecular weight carbohydrates allowing faster analysis.Acetate is a strong competitor for active binding spots resulting inless interaction of the carbohydrates with the ion-exchange groupsof the column (see Fig. 1B). Sodium Acetate is also particularlysuitable for gradient elution. Pulsed amperometric detectors arerelatively insensitive to ionic strength changes of a sodium acetategradient, as long as the sodium hydroxide concentration remainsconstant during the gradient run. High purity grade sodium acetateshould be used for the preparation of the mobile phase (impuritiescan cause large baseline shifts during a gradient run).Pulsed Amperometric Detection (PAD) - DC amperometry is not aparticularly suitable method for the electrochemical detection ofcarbohydrates. The response of the working electrode is quicklyattenuated, as a result of strong adsorption of the oxidation productsonto the working electrode surface.Page : 1/10E1 (Determination)Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comt delayt sampleTime (s)E2 (Oxidation Au)E3 (Reduction Au)Fig. 2. Three-step potential waveform for the pulsed amperometricdetection of Carbohydrates.For that reason Pulsed Amperometric Detection (PAD) with a gold(Au) working electrode has been developed in order to enablefrequent (typically 1 - 2 Hz) renewal of the working electrode (WE)surface [ 1 , 2 ]. The repeating three-step potential waveform isschematically shown in Fig. 2. The three potentials E1, E2 and E3are applied to the Au WE for specified times t1, t2 and t3, respectively.E1 is the actual detection potential. During t1 the carbohydratesare oxidized at the Au electrode surface resulting in aspecific oxidation current. This oxidation current is sampled duringa small time period tsample at the end of t1. The delay time tdelaybefore the start of the actual current measurement assures thatthe contribution of the electrode charging current caused by thepotential steps is small. A large positive potential E2 is applied fora period t2 to achieve anodic formation of surface oxides in orderto clean the electrode surface. During t3 the oxidized electrodesurface is reduced back to a reactive Au surface, the so-calledreactivation step. So within every individual pulse cycle, the carbohydratesignal is measured and the working electrode surfacecleaned and reactivated resulting in a reproducible detector responseover time. Typical settings used in our laboratory for theanalysis of carbohydrates are given in Tabel 1. In this specificexample the total pulse duration is 1000 ms (1 Hz).Detailed information about waveform optimisation for pulsedamperometric detection of carbohydrates can be found in severalpapers published by LaCourse and Johnson [ 3 , 4 ]. The optimumoxidation potential (E1) for the carbohydrates can be obtained bycyclic voltammetry, which gives the current-voltage (I/E) relationshipof the analyte. The cleaning potential (E2) should be highenough and the time duration long enough to remove the carbohydrateoxidation products completely. In alkaline media goldoxide is already formed at E > + 200 mV (vs. Ag/AgCl). At a higherpotential the formation of a metal oxide layer is accelerated and ashorter time setting can be chosen. It is essential that the timeduration (t3) and the magnitude of the reactivation potential (E3) besuch that the metal oxide layer is completely removed. Reductivedissolution already occurs at E3 < 0 mV, but a more negativevoltage speeds up this process. However a too low potential willresult in the chemical reduction of other compounds such as theO2 dissolved in the mobile phase for instance, leading to baselineinstabilities. The S/N ratio can be optimised by adjusting t1 andtsample. In principle the time delay determines the level of the backgroundcurrent. In practise, often a delay time (tdelay) of 100 – 400ms is used, and a sample time of 20 – 200 ms.216_001 #03Page : 2/10THE BRIGHTEST LC-EC APPLICATIONS FORENVIRONMENTAL ANALYSISEVER PLOTTED OUTChloro- and nitrophenols2,4-dinitrophenol (DNP)phenol (P)4-nitrophenol (4-NP)2-methyl-4,6-dinitrophenol (MDNP)2-chlorophenol (2-CP)2-nitrophenol (2-NP)2,4-dimethylphenol (DMP)4-chloro-3-methylphenol (CMP)2,4-dichlorophenol (DCP)2,4,6-trichlorophenol (TCP)pentachlorophenol (PCP)Ketones220_002 #03ANALYSIS OF CARBOHYDRATESANION-EXCHANGE CHROMATOGRAPHY AND PULSED AM-PEROMETRIC DETECTIONConfigurations[A] The ALEXYS 100 'Carbohydrates I' isocratic system(p/n 180.0052)A complete system solution consisting of an ALEXYS 100 LC-ECsystem with He-proof solvent bottles, ion-exchange column,guard column and FLEXCELL.Fig. 3. ALEXYS 100 ‘Carbohydrates I’ isocratic, including column, flow celland LC connection kit.[B] The ALEXYS 100 'Carbohydrates II' gradient system(p/n 180.0054)The high-pressure gradient system, has in addition to systemabove one additional LC 100 pump for HPLC gradients and theOR 100 organiser rack 'dual channel' option (extra pulse damper).Fig. 4. ALEXYS 100 ‘Carbohydrates II’ gradient , including two pumps,column, flow cell and LC connection kit.Materials• HPLC grade water, resistivity > 18 MOhm.cm• Sodium Hydroxide 50 wt% solution, pro-analyse,BOOM, the Netherlands• Sodium Acetate trihydrate, >99%, J.T. Baker, the Netherlands• Helium 5.0, 99.999%, Messer, the Netherlands• Mono- and disaccharides standards, Sigma, USA• Maltodextrin, 96.9%, artificial sweetener, BelgiumMobile phase preparationSodium carbonate is a stronger anion than sodium hydroxide. Theformation of carbonate in the mobile phase is the most commoncause of loss of analyte retention. Carbon dioxide gas present inair will dissolved as CO32- ions in the strong alkaline eluent. Thedissolved carbonate ions will increase the ionic strength of themobile phase resulting in a shortening of the retention times of thecarbohydrate analytes. Therefore, keeping the mobile phase freeof carbonate is one of the key factors towards reproducible carbohydrateanalyses via Anion-Exchange Chromatography.Fig. 5. Schematic representation of mobile phase bottle with Heliumsparging assembly.Take the following precautions to assure carbonate-free mobilephases• Prepare the NaOH mobile phase using a 50% w/w carbonate-freeNaOH stock solution (commercially available)and thoroughly degassed deionised water (≥ 18MΩ). Commercially available NaOH pellets are not acceptablefor eluent preparation, because they are alwayscovered with a thin adsorbed layer of sodium carbonate.• The mobile phase should be stored in plastic containersinstead of glass containers. NaOH is a strong etchingagent and will react with the inner glass wall resulting inthe release of silicates and borates.• Most dioxide dissolved in deionised HPLC grade water(> 18 Mohm-cm) can be removed by degassing the waterin an ultrasonic bath for 10 – 15 minutes, and subsequentsparging with Helium 5.0 gas.• Subsequently add the appropriate amount of 50% w/wNaOH solution to obtain the final eluent. Always pipettethe necessary amount of NaOH from the top part of the50% NaOH stock solution. Any carbon dioxide presentin the solution will precipitate as sodium carbonate tothe bottom of the flask, leaving the top part of the solutionvirtually carbonate free.• High-purity grade sodium acetate should be used for thepreparation of the mobile phase (impurities can causelarge baseline shifts during a gradient run).It is advisable to prepare fresh mobile phase daily. The mobilephase should be sparged continuously with Helium during theanalysis to obtain reproducible retention times.Antec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Connelley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comMethodTable 1LC-EC conditionsFlow rate 1.5 ml/minTemperature 30 °CPhenolsPage : 1/4Page : 3/10ALEXYS AnalyzersAlexys FOOD & 110 BEVERAGEStandardised ALEXYS Analyzers for routine analysis with fluidly running applicationsALEXYS Carbohydrate Analyzer• QC of food products• Food research, phytochemicals• Detect Contaminants and pollutants in beveragesFOOD & BEVERAGEAPPLICATION NOTEANION-EXCHANGE CHROMATOGRAPHY ANDPULSED AMPEROMETRIC DETECTIONThe ALEXYS Carbohydrate Analyzer gradient systemis suitable for the more demanding analysis of complexcarbohydrate mixtures such as oligosaccharides.With this high-pressure gradient analyzer based onpulsed amperometric detection, “fingerprints” ofoligosaccharides and other complex carbohydratemixtures can be recorded. This can serve as a tool forestimating chain length distribution.Flavonoids and phenolsother polyphenolsCarbohydratesVitamins A, C, D, E, and KINTRODUCTIONCarbohydrates not only providethe most easily accessibleenergy source for ourbody, they also play an importantrole in many physiologicalprocesses. Some examplesof these processes areinterce lula recognition,infection processes, andcertain types of cancer. Thedetermination of carbohydratesis of interes to thefood industry and many fieldsin life sciences.Analytes of interest includesimple mono- or disaccharides(such as glucose andsucrose), oligosaccharides(Maltodextrin), polysaccharides(starch, ce lulose) andglycoproteins.Numerous analytical techniquesfor carbohydrate analysishave been developedover the years [6]. The polar,non-volatile, water-solublecarbohydrates are not easilyre-solved. Also detection ofthese non-chromophoriccompounds is di ficult. Carbohydrateshardly have anyUV absorption and thereforeeithe refractive index (RI) orelectrochemical detection(EC) is used for analysis.• Carbohydrates in food industry and fields in life sciences• Analytes of simple mono- or disaccharides• Analytes of oligosaccharides (Maltodextrin)• Analytes of polysaccharides (starch, ce lulose)• Analytes of glycoproteinsDue to the poor detection limits of RI, EC has become the prefered detection method in trace analysis. Anion-ExchangeChromatography at high pH in combination with pulsedamperometric detection (PAD) is nowadays the most sensitiveanalytical method available for carbohydrate analysis [1,2].In this application note we present a simple and sensitive systemsolution for the determination of sugars and oligosaccharides infood products based on the ALEXYS 100 'Carbohydrate' systems.MethodAnion Exchange Chromatography - Under alkaline conditions (pH> 12) carbohydrates can be separated by means of Anion-Exchange Chromatography. Carbohydrates are weak acids witheither completely or partia ly ionised depending on their pKavalue. Due to the extreme alkaline conditions only polymericanion-exchange columns are suitable for carbohydrate separation.The retention time of carbohydrates is inversely co related withpKa value and increases significantly with molecular weight. Theelution order of carbohydrates on such anion-exchange columnsis usually as fo lows: sugar alcohols elute first, fo lowed by mono-,di-, tri-, and higher oligosaccharides.CarbohydratespKa values ranging between 12 and 14. At high pH they wi l beAntec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Conne ley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comGlucoseFructoseLact & SucrGlucoseFructoseGlucoseSucroseLactoseFruct & SucrGlucoseFructoseSucroseLactoseMaltoseLactoseMaltoseMaltoseMaltosePotential (V)ENVIRONMENTALALEXYS Analyzers with fluidly running applicationsALEXYS Phenol Analyzer• Water and Soil samples• Ketones in biodiesel exhaust• Used by CRO labs for routine analysisChlorophenols and nitrophenols are used for industrialand agricultural reasons, thus posing a threat tocontaminating river and drinking water. The MAC(maximum admissible concentration) in the EECcountries for phenols in drinking water is 0.5 μg/l.In the 70’s the US Environmental Protection Agency(EPA) made a list of the eleven most importantphenol contaminants as priority pollutants.PHENOLSIN WATERINTRODUCTIONChlorophenols and nitrophenolsare used in industry andagriculture for several purposes.In the end they may befound in river or drinkingwater. The MAC (maximumadmissible concentration) inthe EEC countries for phenolsin drinking water is 0.5 µg/l. Inthe 70’s the US environmentalprotection agency (EPA)made a list of the eleven mostimportant phenol contaminantsas priority po lutants.The standard EPA method isbased on a concentratingliquid-liquid extraction followedby derivatisation andGC analysis with electroncapture detection.In this application a HPLCmethod for the analysis of the11 EPA phenols in water isdescribed using electrochemicaldetection. Detection limitsare between 25 and 220 ng/l,except for DNP (0.9 µg/l), TCP(0.95 µg/l) and PCP (6 µg/ml).ENVIRONMENTALAPPLICATION NOTE• Chlorophenols in industry and agriculture .• Nitrophe-nols in industry and agriculture• 11 EPA phenols in waterThe detection potential is optimised by constructing I/E relationshipsfor 9 phenols. Due to the poor detection characteristics ofsome phenols, especia ly 2,4 DNP, a working potential of 1200mV vs. Ag/AgCl is used for further experiments. The backgroundcurrent rent is approximately 200 nA.Working electrode contamination was only problematic at highphenol concentrations. The concentrations in samples are in thelow ppb range or lower. A the ppb level no significant contaminationof the working electrode could be measured within one day.One cleaning procedure a the end of each day is su ficient tomaintain reproducible working conditions.Column Spherisorb ODS2, 100x4.6 mm, 3 µmMobile phase 50 mM HAc/NaAc, pH 4.0, 35% acetonitrileSample 100 - 1000 nM phenols, 20 µl injectionAntec Leyden B.V. - Industrieweg 12 - 2382 NV - Zoeterwoude - The Netherlands - info@myantec.com - www.myantec.comAntec (USA) - Suite 110 - 7502 Conne ley Drive - Hanover MD 21076 - USA - info.usa@myantec.com - www.myantec.comProven analyzers to guarantee to guarantee your performance your performance®11
Page 5: ALEXYS AnalyzersConfiguring ALEXYS
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Page 14 and 15: DetectorsPart no.Description171.003
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Page 24 and 25: Organizers and racksBottle rackTool
Page 26 and 27: Flow cellsWhy amperometry?Why amper
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Page 30 and 31: Kits, columns and valvesValves250.0
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Page 37 and 38: THE FINEST LC-EC APPLICATIONS FORFO
Page 39 and 40: DistributorsKOREAYoung Lin Instrume

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