Maximize your Capillary Electrophoresis System GUIDE TO CAPILLARIES R - PDF document

Maximize your Capillary Electrophoresis System GUIDE TO CAPILLARIES, REAGENTS,AND SUPPLIES FOR CE 2 29 5 6 7 8 9 11 12-13 14 15-16 1718-19 20-21 22-23 24-25 26-27 30-31 Table of ContentsCE Method DevelopmentCE Method OptimizationCE Solutions KitsInorganic Anion Solutions KitCation Solutions KitStandard Bare Fused-Silica CapillariesExtended Light Path (Bubble Cell) Bare Fused-Silica CapillariesUniversal Bare Fused-Silica CapillariesPolyvinyl Alcohol (PVA) Coated CapillariesCross-linked and Bonded SIL CapillariesAlignment Interfaces and Capillary CassetteHigh Sensitivity Detection CellCE Standards & ReagentsCE System Start-up and Test KitsInstrument Parts and SuppliesBasic Capillary Electrophoresis Troubleshooting CONTENTS Your Link to Success … CE Capillaries, Reagents,Solutions Kits, and Supplies from Agilent TechnologiesAgilent Technologies, a world leader incapillary electrophoresis (CE) technology, is committed to helping you improve thesolutions, simplifying the use of softwareand instrumentation, and offeringThe Agilent 7100 CE system delivers best-in-class analytical performance, theindustrys broadest selection of detectorsand plug-and-play compatibility with all ofAgilents 6000 Series mass spectrometers. Cap

illary Electrophoresis (CE) offers fastseparations with exceptional efficiency andresolution for charged substances such asbiomolecules, low molecular weight basic oracidic drugs and ions„separations that areoften difficult to achieve with HPLC. CE alsoexcels where sample amounts are very limitedand requires much less buffer than liquid orion chromatography. Used in standalonemode, as the separations component of aCE/MS or as a complementary, orthogonaltechnology to LC, CAPILLARY ELECTROPHORESIS Agilent continues to introduce new CEsolutions kits designed to simplify many of€Inorganic anions€Cations€Organic acids€Forensic anionsThese kits include all you need to beginyour CE analyses, including buffers,capillaries, conditioning solutions, testsamples, methods and detailed descriptions.Each kit is designed to take advantage ofthe automation of the Agilent CE system to make your time in the laboratory moreefficient. All kits are prepared using thesame quality procedures as our buffers and are thoroughly tested and supported.While the kits have been optimized for usewith the Agilent CE system, they may beused with virtually any commercial or home-built CE system. 12345678 1.Formate2.Acetate3.Propionate4.n-Butyrate5.n-Pe

ntanoate6.n-Hexanoate7.n-Heptanoate8.n-Octanoate CE Solutions Kits CE SOLUTION KITS Separation of short-chain carboxylic acids using the Organic Acids Solution Kit. Inorganic Anion Solutions KitThe Inorganic Anion Solutions Kit containsall components needed for the analysis ofcommon inorganic anions such as chloride,bromide, iodide, fluoride, sulfate, andphosphate. Applications include the analysis€Ultra pure water€Waste water€High purity chemicals€Drug formulations€Pulp and paper solutions€Semiconductor solutionsUsing an indirect UV detection systemoptimized for small anions, analyses aresensitive and rapid, and provide analternative to traditional ion chromatography.The kit contains buffer, capillaries, testmixture, and instructions. Separation of common anions.1.Thiosulfate2.Bromide3.Iodide4.Chloride5.Sulfate6.Nitrite7.Nitrate8.Molybdate (VI)9.Azide10.Thiocyanate11.Chlorate12.Fluoride CE SOLUTION KITS Inorganic Anion Solutions Kit Description Part No. Inorganic Anion Solutions Kit5063-6511Inorganic anion buffer250 mL8500-6797Ultra pure CE water500 mL5062-85780.1 N sodium hydroxide250 mL5062-85751.0 N sodium hydroxide250 mL5062-8576Bare fused-silica capillary, 50 m id, 72 cm long2/pkG1600-62211Inorganic anion test

mixtureIncludes 1000 ppm each of fluoride, chloride, bromide, nitrite, sulfate and 2000 ppm phosphate10 mL5062-8524 Cation Solutions KitThe Cation Solutions Kit provides everythingyou need for the analysis of inorganic andlow-molecular-mass organic cations. It isspecially designed for the separation ofalkali metal ions, alkaline-earth metal ionsand alkyl amines in a wide range ofEach kit contains a cation buffer, bare fused silica capillaries, cation standard, CE grade water and a detailed description of the analysis method and most commonapplications, including detection limits andreproducibility data. The Cation Solutions Kitand the separation methods were developedto fit perfectly with the Agilent CE system andto support its high automation capabilities.The methods are very easy to perform andprovide accurate and quantitative analyses. ACCB Cations in coffee and energy drinks.Cation standardCation standard1.Ammonium2.Potassium3.Sodium4.Calcium5.Magnesium CE SOLUTION KITS Cation Solutions Kit Component Part No. Cation Solutions Kit5064-8206Cation buffer250 mL5064-8203Ultra pure CE water500 mL5062-8578Bare fused silica capillary, extended light path bubble factor (3), 50 m id, 56 cm long2/pkG1600-61232Cation test mi

xture25 mL5064-8205 The Organic Acids Solutions Kit is ideal forthe analysis of short alkyl chain carboxylicacids. Employing an indirect UV detectionagent optimized for organic acids, themethodology is simple, sensitive, andprovides accurate quantitative analysis.Suited for the analysis of organic acids in a wide range of matrices, it is especiallyuseful for determination of organic acids in beverages and food. 12345678923456712118910 Organic acids in beer and red wine.Anions in beer1.Chloride2.Sulfate3.Oxalate4.Formate5.Malate6.Citrate7.Succinate8.Pyruvate9.Acetate10.Lactate11.Phosphate12.PyroglutamateAnions in red wine1.Chloride2.Sulfate3.Tartarate4.Malate5.Citrate6.Succinate7.Pyruvate8.Acetate9.Lactate10.Lactate11.Phosphate Organic Acids Solutions Kit Description Part No. Organic Acids Solutions Kit5063-6510Organic acids buffer250 mL8500-6785Ultra pure CE water500 mL5062-85781.0 N sodium hydroxide250 mL5062-8576Bare fused-silica capillary, 75 m id, 72 cm long2/pkG1600-62311Organic acids test mixtureIncludes 1000 ppm each of malate, succinate, and lactate20 mL8500-6900Note: The following parts should be ordered separately for use with the Agilent CE System:Alignment interface for 75 m id capillary (P/N G1600-60310

) for 1600 CE instruments.Alignment interface for 75 m id capillary (P/N G7100-60310) for 7100 CE instruments. CE SOLUTION KITS This highly focused kit was developedspecifically for the analysis of poisonouscompounds, such as cyanide, azide, selenate,arsenate, and arsenite. In cases of poisoning,analytical tools are needed to determine the identity of toxins quickly and accurately.A rapid determination of anionic toxins in beverages using CE with indirect UV detection.Forensicand other anions can be detected within 15 minutes with minimal sample preparation. 123456789101112131415 Analysis of an anion standard with the Forensic Anions Solutions Kit.1.Cyanide2.Chloride3.Nitrite4.Nitrate5.Sulfate6.Azide7.Selenate8.Carbonate9.Fluoride10.Arsenate11.Phosphate12.Glutamate13.Acetate14.Arsenite15.LactateForensic Anions Solutions Kit Description Part No. Forensic Anions Solutions Kit5 x 50 mL5064-8208Basic anion buffer50 mL5064-8209Ultra pure CE water500 mL5062-8578Bare fused-silica capillary, 50 m id, 104 cm long2/pkG1600-64211Inorganic anion test mixtureIncludes 1000 ppm each of fluoride, chloride, bromide, nitrite, sulfate and 2000 ppm phosphate10 mL5062-8524 Forensic Anions Solutions Kit CE SOLUTION KITS Fused-silica capi

llaries are the heartof CE. Pre-aligned capillaries from AgilentTechnologies are designed and optimized for ease of use and reliability. All capillaryends are cut to a smooth, mirror-like finish.In addition, the polyimide outer coating isremoved from the ends. These processesensure minimal sample adsorption and helpmaintain sharp peak shapes. All capillarieshave a pre-made detection "window" and abuilt-in alignment stopper that allows rapidand precise insertion in the alignment 8888 CZE of a tryptic digest of recombinant human growth hormone using a standard fused-silica capillary with 75m internal diameter.Different inner diameters of capillariesneed to use different alignment interfacesto guarantee optimal detection. The colorStandard Bare Fused-Silica Capillaries, 2/pk ID (m) Total Length Effective Length(cm) Part No. 503324.5GreenG1600-6321148.540GreenG1600-6021164.556GreenG1600-6121180.572GreenG1600-62211112.5104GreenG1600-64211753324.5BlueG1600-6331148.540BlueG1600-6031164.556BlueG1600-6131180.572BlueG1600-62311112.5104BlueG1600-643111003324.5GrayG1600-6341148.540GrayG1600-6041164.556GrayG1600-6141180.572GrayG1600-62411112.5104GrayG1600-64411 coding of the capillary and the alignmentinterface allow you to easi

ly match thecorrect interface with the capillary. HELPFUL TIPS Standard Bare Fused-Silica Capillaries CE AND CE/MS CAPILLARIES 10 Electroosmotic flow maintains the "plug" flow in thebubble. Optical slits matched to the zone geometry Analysis of cold medicine ingredients in a standard capillary (25 m id) and an Agilent Extended Light PathCapillary.25 m standard capillaryExtended Light Path (Bubble Cell) Bare Fused-SilicaUse Agilent Technologies extended lightpath capillaries ("bubble" cell capillaries) to improve sensitivity 3- to 5-fold overstandard capillaries. With extended lightpath capillaries, the inner diameter isincreased only at the detection window,offering the sensitivity of a wide innerdiameter capillary and the low currentgeneration of a narrow one.Resolution is not sacrificed when used withmatching optical alignment interfaces fromprocess, the diameter is increased three tofive times, with a manufacturing precisionbetter than 3%. Take advantage of thisprocess to extend the detection pathlengthof 25 m id capillaries to 125 m, 50 m to 150 m, and 75 m to 200 m.Extended Light Path (Bubble Cell) Bare Fused-Silica Capillaries, 2/pk ID (m) Total ength Factor Part No. 2548.5405125BlackG1600-6013264.55651

25BlackG1600-6113280.5725125BlackG1600-621325043.5353150RedG1600-6023348.5403150RedG1600-6023264.5563150RedG1600-6123280.5723150RedG1600-62232112.51043150RedG1600-642327548.5402.7200YellowG1600-6033264.5562.7200YellowG1600-6133280.5722.7200YellowG1600-62332112.51042.7200YellowG1600-64332 HELPFUL TIPS Use narrow 25 and 50 m id "bubble"cell capillaries for highly conductivebuffers without sacrificing sensitivity. CE AND CE/MS CAPILLARIES 11 CZE analysis of a tryptic digest of carbonic anhydrase using a standard capillary (id 50 m) compared with an Agilent Extended Light Path Capillary.50 m ID with 150 m bubbleUniversal Bare Fused-Silica CapillariesUniversal Bare Fused-Silica Capillaries have a window, 75 cm effective length and 363 m od, fitting into any CEinstrument. To cut them to the correct length we recommend using the CE Electroosmotic flow maintains the "plug" flow in the bubble. Optical slits matchedto the zone geometry maintain resolution. Capillary ID (m) Total Length (m) Part No. 205160-2660-5505160-2650-5755160-2644-5Universal Bare Fused-Silica Capillaries ID (m) Total Length (cm) Effective Length (cm) Part No. 2010075190-04315010075190-01317510075190-023110010075190-0331 CE AND CE/MS CAPILLARIES 12

12345 Use of PVA coated capillaries to reduce protein adsorption. 123456789 CZE analysis of basic amines using PVA coated capillaries.Polyvinyl Alcohol (PVA) Coated CapillariesPVA coated capillaries contain apermanently adsorbed layer of polyvinylalcohol. This coating minimizes hydro-phobic and electrostatic solute/wallinteractions and eliminates electroosmoticflow (EOF). Using a proprietary depositionprocess, the PVA coating is stable over awide pH range, even under basic conditions,from pH 2.5 to 9.5. This stability allowsthe use of many common CE buffers.Because the silica surface is covered,many proteins and amines can beanalyzed without the peak tailing foundwith uncoated capillaries. In addition,since EOF is eliminated, cumbersomewashing procedures are unnecessary and migration time reproducibility may Each batch of PVA coated capillaries isrigorously tested by Agilent Technologiesand includes a representative electro-pherogram to assure quality.The color coding of the capillary(alignment stopper) and the alignmentinterfaces allow you to easily combinethe correct interface with the capillary.Capillaries for non-Agilent CE systemswithout color code.PVA coated capillaries can be used for avariety of applications,

including proteinfocusing, and small anion analysiswithout the need for flow-reversal agents in the buffer.1.Cytochrome C2.Lysozyme3.Lactoglobulin A4.Lactoglobulin A5.Lactoglobulin B1.Benzidine2.Aniline3.o-Toluidine4.p-Cl-Aniline5.2-Aminonaphthalene6.4,4-Methylene-bis(2-chloroaniline)7.3,3-Dichlorobenzidine8.2-Amino-5-azotoluene9.2-Methyl-5-nitroanilineUntreated fused-silica capillaryPVA coated capillary CE AND CE/MS CAPILLARIES PVA coating is available in standardcapillaries, or in Agilent Extended Light PathCapillaries ("bubble" cell capillaries) for highsensitivity applications. Both capillary typesare available in longer lengths for use innon-Agilent systems.PVA is also available for use with the highsensitivity detection cell for even furtherimproved HPLC-like sensitivity. In addition,PVA coated capillaries are offered for CE-MS applications. The capillaries aredetection window to allow tandem UV-Visand MS detection for improved samplePVA Coated Capillaries for Non-Agilent CE Systems* ID Total Length Length (cm) BubbleFactor Length (m) Part No. 507160050G160U-6121971603150G160U-6123910056450100G160U-6041971600100G160U-61419*Not compatible with borate buffersNote: When extended pathlength capillaries are used in

non-Agilent systems, loss of resolution may be found ifthe axial slit width is not reduced. In Agilent systems, the alignment interface contains properly matched slits to Analysis of meat proteins by c-IEF using PVA capillaries.PVA Coated Capillaries for Agilent CE Systems* ID Total Length Factor Part No. 5064.556050GreenG1600-6121964.5563150RedG1600-6123912521.5050GreenG1600-672197564.55601200BlueG1600-6831912521.5075BlueG1600-6731910048.5400100GrayG1600-6041964.5560100GrayG1600-61419*Not compatible with borate buffersNote: PVA coated capillaries for CE/MS have a blue alignment stopper matching the blue color code of thealignment interface for MS-UV detection. The alignment stopper of the 50 m id PVA capillary for CE/MS has ablack dot for easy identification. CE AND CE/MS CAPILLARIES 14 Restriction fragment separation (36…2645 bp).bonded polymer coating. This CEP coatingshields the silanol functionality of thecapillary surface and helps prevent sampleadsorption. Additionally, EOF is nearlyeliminated, making the capillary ideal forapplications such as DNA separations withsieving polymer buffers.Elimination of EOF also simplifies analysis of anions and organic acids by direct UVdetection. Without EOF reduction, highly

mobile ions such as nitrate can migrate inthe opposite direction to the slower, longerThe CEP coated capillary is stable from pH 2 to 8. It can be used with boratebuffers, offering a different surfacefunctionality to help alleviate sampleadsorption. Each batch of CEP coatedTechnologies and each capillary includes assure quality. CE AND CE/MS CAPILLARIES CEP Coated Capillaries, 2/pk ID Total Length Length (cm) BubbleFactor Length (m) Part No. 7580.572075G1600-62318 15 Analysis of Allelic ladder with SIL-DNA.Cross-linked and Bonded SIL CapillariesSIL-FC and SIL-DNA Capillaries with Windowsdesigned for CE, which are prepared by cross-linking and bonding a novel,proprietary fluorocarbon (FC) polymer. SIL-FC capillaries are chemically inert,hydrophobic, and stable from pH 2.5-10.These capillaries are a must-have for cIEF, protein, peptide and carbohydrateseparations, as well as replaceable gel CEapplications such as oligonucleotides, DNAfragments, and PCR product separations.SIL-DNA capillaries are also coated withan FC polymer but have a 75 m id toaccommodate the viscosity of entangledpolymer solutions. All SIL capillaries arebatch tested to ensure the highestperformance and reproducibility.SIL-WAX Capillaries w

ithWindowsSIL-WAX features a modified, polyethyleneoxide, hydrophilic coating made through aspecial cross-linking and bonding process.The coating effectively masks active silanolsites, offering exceptional efficiency, resolution,peak shape and reproducibility. The highlystable coating and near-zero EOF of SIL-WAX makes the capillary ideal for CE-MS,and protein and peptide separations frompH 2-5. Analysis of Myoglobin tryptic digest using SIL-WAX. CE AND CE/MS CAPILLARIES SIL-DB coated capillaries are available asSIL-DB-1 and SIL-DB-17. In combinationwith a cellulose based buffer system, SIL-DB coated capillaries have been widelyused in cIEF applications, PCR product andDNA fragment separation, and many otherCE applications which require reduced EOF. SIL-DNA, 199-2602 CE AND CE/MS CAPILLARIES Capillary ID (mm) Length Film Thickness (m) Part No. DB-10.05100.05126-1012DB-10.20100.05126-1013DB-10.10100.10127-1012DB-170.10100.05126-1713DB-170.10100.10127-1712DB-170.20100.10127-1713SIL-FC and SIL-DNA Capillaries with Windows Capillary ID (m) Total Length Part No. SIL-FC5080500.0753/pk194-8111SIL-DNA7565500.0752/pk199-2602SIL-WAX50100750.12/pk196-7203SIL-WAX100100750.12/pk197-7202 17 Alignment Interfaces an

d Capillary CassetteAgilent Technologies alignment interfacesare an integral part of the Agilent diode-array detection (DAD) system. Theseinterfaces contain optical slits which areprecisely matched to the capillary innerdiameter for optimized sensitivity and lineardetection range.In combination with the capillary cassette,exchange, protect the fragile detectionwindow and ensure exact alignment of the window in the detector. Quick-changecassette allows capillary exchange in lessNote:The color code of the alignmentinterface must match the color code of the capillary's built-in alignment stopper.Capillary cassette, G7100-60002Capillary cassette Description Part No. Part No. Capillary cassetteG7100-60002G1600-60002Note: Only use G7100-60002 cassette in G7100 and G1600-60002 cassette in G1600. Never mix cassettes. Description Part No. Part No. for standard50GreenGreenG7100-60210G1600-60210G7100-60310G1600-60310for Agilent Extended25BlackBlackG7100-60150G1600-6015050RedRedG7100-60230G1600-6023075YellowYellowG7100-60330G1600-60330interface for 360 mod capillaries, BlueBlueNote: 75, 100 and 150 m id standard capillaries use the same interface (color blue).PVA coated 50 and 75 m id capillary for CE/MS use the same nonmetall

ic interface with color code blue foruse with standard and extended light path capillaries, and the high sensitivity detector cell. HELPFUL TIPS Cassette and interfaces accept allOptical filter for DAD Description Part No. Part No. Optical filter for DAD260 nm, for DNA analysis with polyacrylamide filled G7100-62700G1600-62700Optical filter for DAD280 nm, for cIEF analysis of proteins, e.g. for antibody charge heterogeneity determination CE AND CE/MS CAPILLARIES High Sensitivity Detection CellThe Agilent high sensitivity detection cell …a technological leap which extendssensitivity by an order of magnitude …provides a solution to sensitivity limitationsoften encountered in CE. This improvementwill substantially increase the utility of CE for impurity analysis of chiral drugs,biologicals, and compounds of environmeinterest, among others.The high linear range allows quantificationof both component in one run. This is useful for allimpurity determinations and is especiallyuseful for determining chiral excess.The high sensitivity detection cell for theAgilent CE system not only improvesdetection sensitivity more than 10-fold over standard capillaries, but also extendslinearity beyond 2000 mAU and providesunsurpassed spect

ral fidelity. Theseimprovements are a result of a proprietarymicromachined design which increases thedetection pathlength from 75 m to 1200m while dramatically reducing stray light . The high sensitivity detection cell has adesign comprised of a fused-silica cell bodyand removable capillaries. The light paththrough the cell is made from black fused-silica which significantly minimizes straylight and defines the aperture for the diode-array spectrometer. In addition, thereflective interior functions as a "lightpipe,"ensuring almost 100% transmission of lightwhich entered the cell. These propertiesresult in enhanced linearity and unsurpassedspectral fidelity with the diode-arraydetector.The Agilent High Sensitivity Detection Cell Dramatically Enhances Sensitivityand Linearity for the Agilent CE Capillary Electrophoresis System INSTRUMENT PARTS AND SUPPLIES Characteristics of the Agilent High Sensitivity Detection Cell€Up to 8-fold increase in signal-to-noise€Detector linearity beyond 2000 mAU foraccurate quantitative analysis€Decoupled design allows replaceablecapillaries and reduced cost operation€Special capillary geometry ensures€Full diode-array spectral capabilities€Design fits all Agilent CE instruments Agilent

high sensitivity detection cell vs. 75 m standard capillary for the CZE separation of naphthalene sulfonicHigh sensitivity cell with 7575 m id standard capillaryCapillary Kits for High Sensitivity Detection Cell Description Effective Length(cm) Part No. 75 m capillary kit with 8.5 cm outlet56G1600-6871672G1600-68715PVA coated 75 m capillary kit with 8.5 cm outlet56G1600-68319High Sensitivity Detection Cell Description Part No. Part No. Includes detection cell, 75 m id inlet capillary (72 cm) and outlet capillary (8.5 cm) pair, capillary cassette, fittings (3 fitting screws with seals, 2 fitting caps), cleaning solution,CE Partner CD-ROMG7100-68723G1600-68723CE cell fitting kitIncludes 3 fitting screws, 2 fitting capsReplacement detection cellG1600-60027Cell cleaning fluid, 1 L5062-8529 INSTRUMENT PARTS AND SUPPLIES The CE/MS Adapter Kit simplifies couplingthe Agilent CE system with MS systemsequipped with an electrospray ionization(ESI) source. Integral to this kit is theCE/MS cassette, which completelythermostats the capillary until it exits the CE system. The cassette offers multiplecapillary paths that vary the capillary length.A method development configuration usesonline diode array detection and MS. Forrapi

d or routine MS analysis, the detectorcan be bypassed to decrease the totalcapillary length and reduce analysis time.the complete Agilent 6000 Series massspectrometers, or virtually any electospray-The CE/MS cassette completely thermostatsthe capillary until it exits the CE system.Methods development configuration usesonline diode array detection (DAD) and MS.For rapid or routine MS analysis the DADcan be by-passed to decrease the totalcapillary length and reduce analysis time.The CE/MS Sprayer Kit includes theelectrospray needle and splitter assembly,which allows the direct connection of the CE instrument with Agilent and otherelectrospray MS systems. The CE/MSSprayer Kit needs the CE/MS Adapter Kit to fully support CE/MS coupling. INSTRUMENT PARTS AND SUPPLIES Kit. For coupling with non-Agilent MS please contact the MS vendor.Deconvoluted mass spectrum of a 40 kDa Antibody conjugate. The assigned structures are based ondeconvoluted mass. Insert depicts the electropherograms for separation of different scFv-B conjugate species asdescribed in Agilent Application Note 5991-4433EN. 029,000 29,500 30,000 30,500 31,000 31,500 32,000 32,500 33,000 33,50030,058.4230,814.0831,610.6132,359.1133,197.460.20.40.60.81.41.62.22.4×

103 Deconvoluted mass (amu)Counts = scFv= Desferal CE with tandem UV-Vis and MS detectionallows the analysis of complex mixtures.Analyte mixtures are separated and thecomponents detected via UV-Vis absorption,allowing preliminary identification based onpeak elution time and UV-Vis spectra, orboth, when compared to a standard. Onlinecoupling to electrospray-ionization massspectrometry (ESI-MS) then revealsunambiguous information on the solute'smolecular weight, and possibly structure.CE/MS Adapter Kit Description Part No. For interfacing the Agilent CE system with a mass spectrometerIncludes parts below, which can be ordered separately*CE/MS interface cassette, metallicG1600-60013CE/MS alignment interface for 360 m od capillaries, nonmetallicG1600-60400CE/MS alignment interface for 360 m od capillaries, nonmetallicG7100-60400Bare fused-silica, 50 m id, 125 cm long, 2/pkG1600-67311*Interfacing the capillary requires an electrospray needle which is not included in this kit INSTRUMENT PARTS AND SUPPLIES CE/MS Sprayer Kit Description Part No. CE/MS Sprayer kit(compatible with Agilent ESI and JetStream MS sources)ES needle assemblyG1607-60041CE-ESI sprayerG1607-60002Splitter assemblyG1607-60000PEEK ferrule, 360 m for CE

/MS Sprayer5022-2141Nut, finger-tight fitting and ferrule2/pk0100-1543Flex loc element2/pk1520-0401Gasket1/pkG1607-20030Ion kit (ammonium acetate)5 x 5 mL CE/MS Capillaries Description Color Code Unit Part No. Bare fused-silica, 50 m id, 125 cm longGreen2/pkG1600-67311PVA coated capillary, 50 m id, 125 cm longGreen1/pkG1600-67219PVA coated capillary, 75 m id, 125 cm longBlue1/pkG1600-67319Bare fused-silica, 50 m id, 125 cm long, UV window without UV-VisGreen2/pkG1600-67312PVA coated capillary, 50 m id, 125 cm long, UV window without UV-VisGreen1/pkG1600-67220 CE Standards & ReagentsPremade buffers help eliminate the time-consuming buffer preparation process. AllAgilent buffers and reagents are designedto meet the stringent demands of CE.Manufactured under GLP/GMP conditionsin ISO 9001 facilities, each is shipped withassay information and verification of purity.Chemicals are all electrophoresis grade,with nearly all ionic and organic impuritiesremoved. Solutions are prepared underClass 10 clean room conditions andprefiltered through 0.2 m filters to ensureremoval of particulates. Superior qualityto-bottle and batch-to-batch.In addition to a set of kit buffers, which are specially designed for dedicatedapplication

s, Agilent offers a series of basicCZE buffers covering a broad pH range. buffers for protein analysis and for MicellarElectrokinetic Chromatography (MEKC).Cleaning and conditioning solutionscomplete the offering. Agilent Technologies' Capillary Electrophoresisprogram includes premade buffers designed toreduce your time at the laboratory bench.Capillary Conditioning Solutions Description Volume (mL) Part No. 0.1 N sodium hydroxide250 mL5062-85751.0 N sodium hydroxide250 mL5062-85760.1 N phosphoric acid250 mL5062-8577Ultra Pure CE Water Description Volume (mL) Part No. Ultra pure CE water5005062-8578 INSTRUMENT PARTS AND SUPPLIES 23 400700 The total fluorimetry spectrum of the 50 mM borate buffer pH 9.2 verifies thatthe solution is free of fluorescence-active impurities (1 and 2 = Rayleigh straylight of zero and first order, 3 = Raman stray light). mAUmin101020305086420 CZE analysis of a peptide mixture using pre-made 50 mM sodium phosphatebuffer, pH 2.5. Description Volume (mL) Part No. Plating bath analysis buffer250 mL5064-8236MEKC Buffers for Neutral and Charged Analytes Description Volume (mL) Part No. 50 mM sodium tetraborate, 100 mM sodium dodecyl sulfate buffer, pH 9.3*250 mL5062-8574*Dilute with 50 mM sodium t

etraborate, pH 9.3 (P/N 5062-8573) to reduce SDS concentration without affectingthe tetraborate composition or pH.CZE Buffers for Proteins Description Volume (mL) Part No. 50 mM phosphate, 0.05% hydroxyethyl cellulose buffer, pH 2.5250 mL8500-6786150 mM phosphate, 200 mM ammonium sulfate buffer, pH 7.0250 mL8500-6787CZE Buffers for Charged Analytes Description Volume (mL) Part No. 50 mM sodium phosphate buffer, pH 2.5250 mL5062-857150 mM sodium phosphate buffer, pH 7.0250 mL5062-857250 mM sodium tetraborate buffer, pH 9.3250 mL5062-857320 mM sodium tetraborate buffer, pH 9.3100 mL8500-6782 INSTRUMENT PARTS AND SUPPLIES CE System Start-up and Test KitsChemical test kits and validation packagesare available to help comply with regulatoryand quality standards. The InstallationQualification (IQ) Chemical Kit andHardware Start-Up Kits, which are shippedwith new instruments, are useful for rapidlyverifying system functionality. For rigoroustesting, the Operational Qualification(OQ)/Performance Verification (PV) Kit canbe used to verify DAD noise, drift, linearity,wavelength accuracy and replenishmentfunctionality. The OQ/PV kit is only part of the validation services available fromAgilent Technologies. When implemented by q

ualified Agilent personnel, our servicepackages can be used to help validate yourAgilent CE system. INSTRUMENT PARTS AND SUPPLIES Temperature stabilityWavelength accuracyVoltage stabilityInjector reproducibility and linearityReplenishment functionality 25 IQ and OQ/PV test method. IQ and OQ/PV test method Sample:p-HydroxyacetophenoneBuffer:20 mM Borate pH 9.2 Capillary:I = 40 cm, L = 48.5 cm, 50 m idInjection:250 mbar*sVoltage:30 kVTemperature:20 °CDetector:192/4 nmCE System Start-up and Test Kits Description Part No. CE Installation Qualification (IQ) KitIncludes buffer (20 mM borate, pH 9.3, 100 mL), test sample (4-(hydroxy)-acetophenone, 2 mL), capillary conditioning solution (0.1 N sodium hydroxide, CE Operational Qualification Performance Verification (OQ/PV) Chemical KitIncludes buffer (20 mM borate, pH 9.3, 100 mL), test samples (0.1, 0.5, 1.0, and 5.0 mM 4-(hydroxy)-acetophenone, 2 mL ea.), capillary conditioning solution(0.1 N sodium hydroxide, 100 mL), test capillary (L 48.5 cm, I 40 cm, id 50 m). Note: Method is supported for G1600 only.CE OQ/PV Chemicals Only KitIncludes buffer (20 mM borate, pH 9.3, 100 mL), test samples (0.1, 0.5, 1.0, and 5.0 mM 4-(hydroxy)-acetophenone, 2 mL) INSTRUMENT PARTS AND SUP

PLIES Vials and Caps for CE Description Part No. Crimp/snap top vial, 1 mL, Polypropylene, crimp/snap top100/pk5182-0567Clear, wide opening crimp/snap top vial, 2 mL100/pk5182-9697Amber, wide opening crimp/snap top vial, write-on spot, 2 mL100/pk5183-4619Crimp/snap top vial, 250 L1000/pk9301-0978Snap caps PEO (polyethylene olefin for chemical resistance)100/pk5181-1507Snap caps PEO (polyethylene olefin for chemical resistance)500/pk5181-1513Snap caps PUR (polyurethane for resealing)*100/pk5181-1512Snap caps PUR (polyurethane for resealing)*500/pk5042-6491*PUR caps are recommended to help prevent sample or buffer evaporation even after multiple injections. INSTRUMENT PARTS AND SUPPLIES Description Part No. Long-life HiS Deuterium lamp (8-pin) with RFID tag5190-0917Deuterium lamp2140-0585Electrode assembly, standard (for G1600 only)G1600-60007Electrode assembly, short (for G1600 only)G1600-60033Electrode assembly, standard (for G7100 only)G7100-60007Electrode assembly, short (for G7100 only)G7100-60033Electrode O-ring, silicone5/pk5062-8544Electrolyte bottle, 500 mL9300-1748Electrolyte bottle, 100 mL5042-6478Electrolyte bottle cap9300-1747Bottle sealing O-ring0905-1163Glass filter, solvent inlet, 20 m5041-2168Filter f

rit adapter, 3 mm4/pk5062-8517Bottle cap plugG1600-23223Air filter, 5 m3150-0619Pre-puncherG1600-67201Screws for pre-puncher/insulation plate holding10/pkG1600-62402 Snap caps, polyurethane, 5181-1512, 5042-6491 Electrode assembly, standard (for G1600 only), Electrode O-ring, silicone, 5062-8544 Electrolyte bottle, 500 mL, 9300-1748 Filter frit adapters, 5062-8517 Air filter, 5 m, 3150-0619 Pre-puncher, G1600-67201 Screws for pre-puncher/insulation plate Window Etching ToolThe window etching tool is designed for fast, convenient and reproducible preparation ofdetection windows on fused-silica capillaries. The polyimide coating is removed withoutdestroying the inner polymeric coating. The tool contains three glass blocks with finegrooves, precisely controlling the size of the windows. Part No. CE accessory kitIncludes electrode tool, screwdriver, fuses, air filter, glass frit, vials and caps alignment interfaces (red and green) standard and 50 m id capillaries: Rack for 12 mm, 2 mL vials, holds 50 vials per rack, 5/pk9301-0722CE column cutter5183-4669Diamond blade replacement kit for CE column cutter5183-4670Capillary tubing cutter, 4/pk5181-8836 Description Part No. Window etching tool, 3/pk590-3003 INSTRUMENT PART

S AND SUPPLIES Vial rack, 9301-0722 CE column cutter, 5183-4669 Window etching tool, 590-3003 28 Dissolved in water, diluted bufferor other minimally conductiveDetermine the analyte's chargeDo you know the sample'sPerform CZE experiments using the following buffers:€25 mM phosphate (pH 2.5)€25 mM borate (pH 9.3)CationicAnionicNeutralInitial run:Initial run:Initial run:€25 mM phosphate€25 mM borateCheck concentration by flow-injection analysisIf sample is too dilute:1. use high sensitivity cell2. concentrate sample3. use electrokinetic injectionIf the sample contains high saltconcentration desalt or increasethe buffer concentrationNOYES CE Method DevelopmentBefore attempting to develop a CE method,gather information about the particularcompounds you want to analyze. Helpfulinformation is available from CE instrumenthandbooks, and user organizations, whichyou can find on the Internet. Knowing thesolubility of the compound and finding theright pH value to maintain a stable chargeduring separation is key. There are already a number of standard buffers andexperiments. These will point you in theright direction to create a robust, optimizedapplication method. CE METHOD DEVELOPMENT €25 mM borate and 25 mMsodium dodecyl sulfa

te (pH 9.3)€25 mM borate and 25 mM sodiumdodecyl sulfate (pH 9.3) 29 Optimize the separation forMEKC conditionsCorrect number of peaks? Is the peak shape good?Is the resolution sufficient?Correct number of peaks? Is the peak shape good?Is the resolution sufficient?Correct number of peaks? Is the peak shape good? Is the resolution sufficient?Increase the sodium dodecylsulfate concentration from 25 mM to100 mM in 25 mMincrements and/or decrease the pH to 7 or 8modifier such as acetonitrileAre the separation time and€Injection€Capillary length€Capillary inner diameter€Capillary voltage€Capillary temperatureValidate the methodDetermine method stability,ruggedness and reproducibilityDoes the method meet theOptimize the separation for (in the following order):€Optimize the pH€Increase the bufferconcentrations in 25 mM€Change the buffer ion€If peaks are tailing, add abuffer modifier such asNO (CZE)NO (MEKC) CE Method OptimizationWhether you wish to separate charged compounds via capillary zoneelectrophoresis (CZE) or separate polar toneutral compounds via Micellar electro-kinetic chromatography (MEKC), you willneed to take steps to optimize the processto take full advantage of the high resolutionthat capillary electrophoresi

s offers. Here are some basic steps for successfullyoptimizing CZE and MEKC methods. For more details, please see our primer, High-Performance Capillarynumber 5990-3777EN). CE METHOD OPTIMIZATION 30 Possible Cause Solution(s) Unstable CurrentVariable or no currentAir bubble formed in capillaryFlush capillary, ramp voltage to limit initial heating, and/or degas buffers.Clogged capillaryFlush capillary with absorbing solution (such as NaOH). A "step" on the baseline should be observed when viewing the online signal at 200 nm. If still plugged, flush manually with syringe or high pressure gas.Broken capillaryReplace capillary.No or incorrect solution in buffer vialsFill/change buffer vials.Large volume injectionNormal situation. Current should stabilize during analysis.Unstable BaselineSpikes in baselinePrecipitates in bufferFilter buffer through 0.2 or 0.45 m filter.Micro air bubbles in bufferDegas buffer by ultrasonication or vacuum.Precipitation of sampleVerify that sample components are sufficiently soluble in buffer.Noisy baselineOptical slit in capillary interface is occludedClean slit with methanol or water. View under magnifier.Aging deuterium lampUse DAD test to measure lamp output and time-on. Replace if nece

ssary.Data acquisition rate too highDetermine peak width and decrease acquisition rate if Improper reference wavelengthAcquire UV spectrum during analysis. Use lowest wavelength possible without impinging where sample absorbs. Also use wide bandwidth.Buffer absorbs at detection wavelengthUse minimally UV-absorbing buffers such as phosphate and borate, especially below 210 nm.Drifting baselineImproper capillary alignmentRe-seat capillary cartridge in detector block.Unequilibrated temperatureAllow 10-20 minutes for equilibration after opening top cover.Lamp recently ignitedAllow 15-30 minutes for equilibration after igniting lamp.Poor Peak EfficiencyBroad peaksSample overloadingDecrease sample injection or concentration.Excessive Joule heatingReduce voltage, buffer conductivity, or capillary id.Skewed peaksMismatched sample buffer ion mobilitiesMatch mobilities or increase difference between buffer and sample conductivity.Sample overloadingDecrease sample injection or concentration.Tailing peaksAdsorption to capillary wallUse pH extremes, high buffer concentrations, polymer additives, or coated capillary.Basic Capillary Electrophoresis Troubleshooting TROUBLESHOOTING 31 Possible Cause Solution(s) Poor Migration Time Rep

roducibilityAdsorption to capillary Changes in EOF caused by buffer (especially phosphates and detergents) or sample adsorptionCondition capillary and allow sufficient equilibration time. Replace capillary.Caused by conditioning capillary at high (or low) pH and employing a low (or high) pH runnning bufferAvoid pH differences. Allow sufficient equilibration time.Changes in buffer pH changes due to electrolysisReplenish buffer.Buffer evaporationTightly cap buffer vials and reduce carousel temperature.Conditioning solution waste flushed into outlet reservoirUse separate vial to collect waste.Conditioning solution carried over into buffer vialFirst dip capillary in separate buffer or water vial.Buffer reservoirs not Generation of laminar flowLevel liquid in reservoirs. If not replenishing buffer, do not use inlet vial for flushing capillary.of capillary batchesDifferent wall charge and variations in EOFMeasure EOF and normalize.Temperature changesChanges in viscosity and EOFUse system with capillary thermostatting.Poor Peak Area ReproducibilitySudden application of Heating, thermal expansion of buffer, and expulsion Ramp separation voltage or inject buffer plug after Sample evaporationIncreasing sample concentration and

peak areaCap vials and/or reduce temperature of sample carousel.Instrumental limitationsSystem rise time significant proportion of injection timeIncrease injection time.Poor Peak Area ReproducibilitySample carry-overExtraneous injectionUse capillary with flat, smooth injection end. Remove polyimide from end of capillary.Zero-injection caused by simply dipping the capillary in the sampleExtraneous injectionCannot be totally eliminated. Increase injection amount to minimize effect.Sample adsorption to capillary wallsDistorted peak shape (tailing)Change buffer pH. Increase buffer concentration.Use additive such as cellulose or coated capillary.Low signal-to-noise ratioIntegration errorsOptimize integration parameters. Increase sample concentration. Use peak height.Temperature changes of capillary environmentChanges in viscosity and injection amountUse system with capillary thermostatting. TROUBLESHOOTING Learn more:www.agilent.com/chem/ceBuy online:www.chem.agilent.com/storeFind an Agilent customer center in your country:www.agilent.com/chem/contactusU.S. and Canada1-800-227-9770Europeinfo_agilent@agilent.comAsia Paci“cinquiry_lsca@agilent.com© Agilent Technologies, Inc. 2009-2015Printed in the USA, March 1, 20155991-562