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Agilent generalCE
Agilent generalCE
Maximize your CE and CEC System and experience greater laboratory uptime
Your Link to Success Agilent Technologies Capillary Electrophoresis Capillaries, Reagents, Solutions Kits, and Supplies
Agilent Technologies, a world leader in capillary electrophoresis (CE) technology, is committed to helping you improve the quality of analyses by offering application solutions, simplifying the use of software and instrumentation, and offering unsurpassed technical support. Capillary Electrophoresis (CE) offers fast separations with exceptional efficiency and resolution for charged substances such as biomolecules, low molecular weight basic or acidic drugs and ionsseparations that are often difficult to achieve with HPLC. CE also excels where sample amounts are very limited and requires much less buffer than liquid or ion chromatography. Used in standalone mode, as the separations component of a CE/MS or as a complementary, orthogonal technology to LC, The Agilent 7100 CE system delivers bestin-class analytical performance, the industrys broadest selection of detectors and plug-and-play compatibility with all of Agilents 6000 Series mass spectrometers.
Table of Contents
CE Method Development CE Method Optimization CE Solution Kits Inorganic Anion Solutions Kit Cation Solutions Kit Organic Acids Solutions Kit Forensic Anions Solutions Kit PAGE Solutions Kits for High Resolution DNA Fragments Analysis Standard Bare Fused-Silica Capillaries Extended Light Path (Bubble Cell) Bare Fused-Silica Capillaries Universal Bare Fused-Silica Capillaries Poly Vinyl Alcohol (PVA) Coated Capillaries CEP Coated Capillaries Cross-linked and Bonded SIL Capillaries Capillary Electrochromatography (CEC) Capillaries Alignment Interfaces and Capillary Cassette High Sensitivity Detection Cell CE/MS Accessories CE Standards & Reagents CE System Start-up and Test Kits Instrument Parts and Supplies Basic Capillary Electrophoresis Troubleshooting 4 5 6 7 8 9 10 10 12 13 14 15 17 17-18 19 20 21-22 23 24-25 26-27 28-29 30-31
CE Method Development
Sample dissolved in water, diluted buffer or other minimally conductive matrix
NO
Determine the analyte's charge
YES
Perform CZE experiments using the following buffers: 25 mM phosphate (pH 2.5) 25 mM borate (pH 9.3) 25 mM borate 25 mM sodium dodecl sulfate (pH 9.3) Cationic Anionic Neutral
NO
Any peaks
YES
If sample is too dilute: 1. use High Sensitivity Cell 2. concentrate sample 3. use electrokinetic injection If the sample contains high salt concentration desalt or increase the buffer concentration
CE Method Optimization
Optimize the separation for MEKC conditions Optimize the separation for CZE conditions
Correct number of peaks? Is the peak shape good? Is the resolution sufficient?
YES
YES
Correct number of peaks? Is the peak shape good? Is the resolution sufficient?
NO
YES
NO
Increase the sodium dodecyl sulfate concentration from 25 mM to100 mM in 25 mM increments and/or decrease the pH to 7 or 8
NO
Adjust variables (in the following order): Optimize the pH Increase the buffer concentrations in 25 mM increments Change the buffer ion If peaks are tailing, add a buffer modifier such as hydroxymethylcellulose or use a coated capillary
Optimize Injection Capillary length Capillary inner diameter Capillary voltage Capillary temperature
Correct number of peaks? Is the peak shape good? Is the resolution sufficient? Validate the method Determine method stability, ruggedness and reproducibility
NO (MEKC)
Add 5-30 % of an organic modifier such as acetonitrile Does the method meet the analysis criteria?
NO (CZE)
YES
Method completed 5
CE Solution Kits
Agilent continues to introduce new CE solutions kits designed to simplify many of your applications: Inorganic anions Cations Organic acids Forensic anions Page kits for high resolution DNA fragment analysis These kits include all you need to begin your CE analyses, including buffers, capillaries, conditioning solutions, test samples, methods and detailed descriptions. Each kit is designed to take advantage of the automation of the Agilent CE system to make your time in the laboratory more efficient. All kits are prepared using the same quality procedures as our buffers and are thoroughly tested and supported.
1 2 34 5 6 7 8
1. 2. 3. 4. 5. 6. 7. 8.
Separation of short-chain carboxylic acids using the Organic Acids Solutions Kit
While the kits have been optimized for use with the Agilent CE system, they may be used with virtually any commercial or home-built CE system.
Thiosulfate Bromide Iodide Chloride Sulfate Nitrite Nitrate Molybdate (VI) Azide Thiocyanate Chlorate Fluoride
Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for standard 50 m ID capillary (P/N G1600-60210)
1. 2. 3. 4. 5.
Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for 50 m ID extended light path capillary (P/N G1600-60230).
Chloride Sulfate Oxalate Formate Malate Citrate Succinate Pyruvate Acetate Lactate Phosphate Pyroglutamate
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Chloride Sulfate Oxalate Formate Malate Citrate Succinate Pyruvate Acetate Lactate Phosphate
Anions in beer
10 3 6 2 5 7 3 4 8 11 1 2 6 4 5 7 9 8 9
Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for 75 m ID capillary (P/N G1600-60310)
9 10 11 12 67 8 12 13 14 15
Note: The following part should be ordered separately for use with the Agilent CE System: Alignment interface for standard 50 m ID capillary (P/N G1600-60210)
10
PAGE capillaries are available in three different pore sizes. The size of the molecular sieving pores is controlled by the monomer concentration (% T) and the degree of polymer cross-linking (% C). Gels with higher %T and %C values have smaller pores and are, therefore, more effective at resolving smaller smaller molecules PAGE10 (10 % T, O % C) capillaries provide high resolution capabilities for separation of antisense therapeutic agents, primers and probes as well as nucleotides. PAGE-5 (5 %T, 5 %C) allows single base resolution of oligonucleotides [pd(A)] ranging from 20-150 bases. For your convenience, PAGE capillaries and PAGE buffers can be purchased together or separately. To achieve the highest reproducibility and provide optimal longevity, use PAGE buffer with PAGE capillaries.
75
192-5211
75
192-3211
75
191-5211
Note: The PAGE capillaries are not pre-aligned for the G1600A CE and G7100 CE systems. To cut them to the correct length use the CE column cutter (P/N 5183-4669). To create Detection Window, use the Window Etching Tool (P/N 590-3003).
11
8888
CZE of a tryptic digest of recombinant human growth hormone using a standard fused silica capillary with 75 m internal diameter
75
100
Helpful hint:
Different inner diameters of capillaries need to use different alignment interfaces to guarantee optimal detection. The color coding of the capillary and the alignment interface allow you to easily match the correct interface with the capillary.
12
Electroosmotic flow maintains the "plug" flow in the bubble. Optical slits matched to the zone geometry maintain resolution
25 m standard capillary
Use Agilent Technologies extended light path capillaries ("bubble" cell capillaries) to improve sensitivity 3- to 5-fold over standard capillaries. With extended light path capillaries the inner diameter is increased only at the detection window, offering the sensitivity of a wide inner diameter capillary and the low current generation of a narrow one. Resolution is not sacrificed when used with Agilent Technologies' matching optical alignment interfaces. Through a computer-controlled, proprietary process the diameter is increased three to five times with a manufacturing precision better than 3%. Take advantage of this process to extend the detection pathlength of 25 m ID capillaries to 125 m, 50 m to 150 m, and 75 m to 200 m.
Analysis of cold medicine ingredients in a standard capillary (id 25 m) and an Agilent Extended Light Path Capillary
25
50
75
Black Black Black Red Red Red Red Red Yellow Yellow Yellow Yellow
G1600-60132 G1600-61132 G1600-62132 G1600-60233 G1600-60232 G1600-61232 G1600-62232 G1600-64232 G1600-60332 G1600-61332 G1600-62332 G1600-64332
Helpful hint:
Use narrow 25 and 50 m id "bubble" cell capillaries for highly conductive buffers without sacrificing sensitivity
13
50 m ID
Electroosmotic flow maintains the "plug" flow in the bubble. Optical slits matched to the zone geometry maintain resolution
CZE analysis of a tryptic digest of carbonic anhydrase using a standard capillary (id 50 m) compared with an Agilent Extended Light Path Capillary
14
4 3
1. 2. 3. 4. 5. 6. 7. 8. 9.
15
The PVA coating is available in standard capillaries, or in Agilent Extended Light Path Capillaries ("bubble" cell capillaries for high sensitivity applications. Both capillary types are available in greater lengths for use in non-Agilent systems.
50
Now the PVA is also available for use with the High Sensitivity Detection Cell for even further improved HPLC-like sensitivity. In addition PVA coated capillaries are offered fro CE-MS applications. The capillaries are provided with normally positioned detection window to allow tandem UV-Vis and MS detection for improved sample identification.
75 100
0 3 0 0 0 0 0
*Not compatible with Borate buffers Note: The PVA capillaries for CE/MS have a blue alignment stopper matching the blue color code of the alignment interface for MS-UV Det. The alignment stopper of the 50 m ID PVA capillary for CE/MS has a black dot for easy identification.
*Not compatible with Borate buffers Note: When extended pathlength capillaries are used in non-Agilent systems, loss of resolution may be found if the axial slit width is not reduced. In Agilent systems, the alignment interface contains properly matched slits to maintain resolution.
Goat Lamb
16
The CEP coated capillary is stable from pH 2 to 8. It can be used with borate buffers offering a different surface functionality to help alleviate sample adsorption. Each batch of CEP coated capillaries is rigidly tested by Agilent Technologies and each capillary includes a representative electropherogram to assure quality.
17
50 75 50 100
18
Isocratic conditions I
Isocratic conditions II
1. 2. 3. 4. 5. 6.
Chlorothiazide Hydrochlorothiazide Chlorthalidone Hydroflumethiazide Bendroflumothiazide Bumetanide Buffer: 50 mmol/Na2HPO4, pH 2.5 60:20:20 v/v, MeCN/, Buffer/H20
Capillary Electrochromatography of diuretic test mixture (courtesy of Dr. Melvin Euerby, Astra Charnwood, UK)
Use CEC to improve resolution of solutes which are difficult to resolve by HPLC, for hydrophobic solutes which cannot be solubilized in MEKC buffers, or for reduced sample and solvent consumption compared to HPLC.
*The color coding of the capillary (alignment stopper) and the interface allows you to easily combine the correct alignment interface with the capillary.
2 34 5 6 1 7 8 9 10 11
12
Thiourea Methylparaben Ethylparaben Ptopylparaben Butylparaben Pentylparaben Naphthalene Biphenyl Fluorene Phenanthrene Anthracene Fluoranthene
CEC capillaries require an Agilent CE system with external gas supply capabilities.
19
Alignment Interfaces
Description Alignment interface for standard capillary ID (m) 50 75 100 150 25 50 75 Color Code Green Blue Gray Brown Black Red Yellow Blue Corresponding Capillary Green Blue Gray Brown Black Red Yellow Blue Gray G7100 CE Part No. G1600 CE Part No.
Alignment interface for Agilent Extended Light Path capillaries CE/MS alignment interface for 360 m OD capillaries, nonmetallic
Note: 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 nonmetallic interface with color code blue for use with standard and extended light path capillaries, and the high sensitivity detector cell.
Capillary cassette
Description Capillary cassette
Alignment Interfaces
G7100-60002 G1600-60002
Note: Only use G71000-60002 cassette in G7100 and G1600-6002 cassette in G1600. Never mix cassettes.
Helpful hint:
Optical filter for DAD G7100-62700 G1600-62700 260 nm, for DNA analysis with polyacrylamide filled capillaries and oligonucleotide analysis
The cassette and interfaces accept all commercially available capillaries (~365 m od).
20
21
Agilent High Sensitivity Detection Cell vs. 75 m Standard Capillary for the CZE separation of naphthalene sulfonic acids
22
CE/MS Accessories
The CE/MS adapter kit simplifies coupling the Agilent CE system with MS systems equipped with an electrospray ionization (ESI) source. Integral to this kit is the CE/MS cassette which completely thermostats the capillary until it exits the CE system. The cassette offers multiple capillary paths which vary the capillary length. A methods development configuration uses on-line diode array detection and MS. For rapid or routine MS analysis the detector can be bypassed to decrease the total capillary length and reduce analysis time. The CE/MS adapter kit can be used with the complete Agilent 6000 Series mass spectrometers, or virtually any electospray-MS platform.
1. 2. 3. 4. Leu-Enkephalin, MW 555.6 Met-Enkephalin, MW 573.7 VYV, MW 379.4 Angiotensin II, MW 1046.2 2 1 3 4
UV
MS-TIC (scan)
The CE-MS cassette completely thermostats the capillary until it exits the CE system. Methods development configuration uses online diode array detection (DAD) and MS. For rapid or routine MS analysis the DAD can be by-passed to decrease the total capillary length and reduce analysis time.
The CE-ESI-MS Nebulizer Kit includes the electrospray needle and splitter assembly which allows the direct connection of the CE instrument with Agilent and other electrospray MS systems. The CE-ESI-MS Nebulizer Kit needs the CE-MS Adapter Kit for fully supporting the CE-MS coupling.
Interfacing the capillary requires an electospray needle which is not included in this kit but in the CEESI- MS Nebulizer Kit. For coupling with non-Agilent MS please contact the MS vendor
23
The CE with tandem UV-Vis and MS detection allows the analysis of complex mixtures. Analyte mixtures are separated and the components detected via UV-Vis absorption, allowing preliminary identification based on peak elution time and/or UV-Vis spectra when compared to a standard. The on-line coupling to electrospray-ionization mass spectrometry (ESI-MS) then reveals unambiguous information on the solute's molecular weight and possibly also structure.
CE/MS Capillaries
Description Bare fused-silica, 50 m ID, 125 cm long PVA coated capillary, 50 m ID, 125 cm long PVA coated capillary, 75 m ID, 125 cm long Color Code Green Green Blue Unit 2/pk 1/pk 1/pk Part No. G1600-67311 G1600-67219 G1600-67319
Agilent Technologies' Capillary Electrophoresis program includes premade buffers designed to reduce your time at the laboratory bench
24
Description 0.1 N sodium hydroxide 1.0 N sodium hydroxide 0.1 N phosphoric acid
Volume (mL) Part No. 250 250 250 5062-8575 5062-8576 5062-8577
The total fluorimetry spectrum of the 50 mM borate buffer pH 9.2 1 verifies that the solution is free of fluorescence-active impurities (1 and 2 = Rayleigh stray light of zero and first order, 3 = Raman stray light)
mAU 50 40 30 20 10 0 2 4 6 8 10 min
*Dilute with 50 mM sodium tetraborate, pH 9.3 (P/N 5062-8573) to reduce SDS concentration without affecting the tetraborate composition or pH.
25
Temperature stability
Voltage stability
Replenishment functionality
26
27
*PUR caps are recommended to help prevent sample or buffer evaporation even after multiple injections
Instrument Supplies
Description Long life Deuterium lamp (8-pin) with RFID tag (for G7100) Deuterium lamp (for G1600) Electrode assembly, standard (for G1600 only) Electrode assembly, short (for G1600 only) Electrode assembly, standard (for G7100 only) Electrode assembly, short (for G7100 only) Electrode O-ring, silicone Electrolyte bottle, 500 mL Electrolyte bottle, 100 mL (for G7100 only) Electrolyte bottle cap Bottle sealing O-ring Glass filter, solvent inlet, 20 m Filter frit adapter, 3 mm Bottle cap plug Air filter, 5 m Pre-puncher Screws for pre-puncher/insulation plate holding Unit Part No. 5190-0917 2140-0585 G1600-60007 G1600-60033 G7100-60007 G7100-60033 5062-8544 9300-1748 5042-6478 9300-1747 0905-1163 5041-2168 5062-8517 G1600-23223 3150-0619 G1600-67201 G1600-62402
5/pk
4/pk
10/pk
28
Accessories
Description CE accessory kit Includes electrode tool, screwdriver, fuses, air filter, glass frit, vials and caps. Includes 50 m ID capillaries: L 64,5, Standard: L 64.5, Extended Light Path; L: 48.5, Standard, and alignment interfaces (red and green) Rack for 12 mm, 2 mL vials, holds 50 vials per rack, 5/pk CE column cutter Diamond blade replacement kit for CE column cutter Capillary tubing cutter, 4/pk
5183-4669
29
Broken capillary No or incorrect solution in buffer vials Large volume injection Unstable Baseline Spikes in baseline Precipitates in buffer Micro air bubbles in buffer Precipitation of sample Noisy baseline Optical slit in capillary interface is occluded. Aging deuterium lamp Data acquisition rate too high Improper reference wavelength
Filter buffer through 0.2 or 0.45 m filter. Degas buffer by ultrasonication or vacuum. Verify that sample components are sufficiently soluble in buffer. Clean slit with methanol or water. View under magnifier. Use DAD test to measure lamp output and time-on. Replace if necessary. Determine peak width and decrease acquisition rate if appropriate. Acquire UV spectrum during analysis. Use lowest wavelength possible without impinging where sample absorbs. Also use wide bandwidth. Use minimally UV-absorbing buffers such as phosphate and borate, especially bewlow 210 nm. Re-seat capillary cartridge in detector block. Allow 10-20 minutes for equilibration after opening top cover. Allow 15-30 minutes for equilibration after igniting lamp.
Buffer absorbs at detection wavelength Drifting baselne Improper capillary alignment Unequilibrated temperature Lamp recently ignited Poor Peak Efficiency Broad peaks Skewed peaks Sample overloading Excessive Joule heating Mismatched sample buffer ion mobilities Sample overloading Tailing peaks Adsorption to capillary wall
Decrease sample injection or concentration. Reduce voltage, buffer conductivity, or capillary id. Match mobilities or increase difference between buffer and sample conductivity. Decrease samplle injection or concentration. Use pH extremes, high buffer concentrations, polymer additives, or coated capillary.
30
Symptom
Possible Cause
Solution(s)
Poor Migration Time Reproducibility Adsorption to capillary walls Hysteresis of wall charge Changes in buffer composition Changes in EOF caused by buffer (especially phosphates and detergents) or sample adsorption Caused by conditioning capillary at high (or low) pH and employing a low (or high) pH runnning buffer pH changes due to electrolysis Buffer evaporation Conditioning solution waste flushed into outlet reservoir Conditioning solutiion carried over into buffer vial Buffer reservoirs not level Generation of laminar flow Condition capillary and allow sufficient equilibration time. Replace capillary. Avoid pH differences. Allows sufficient equilibration time. Replenish buffer. Tightly cap buffer vials and reduce carousel temperature. Use separate vial to collect waste. First dip capillary in separate buffer or water vial. Level liquid in reservoirs. If not replenishing buffer, do not use inlet vial for flushing capillary. Measure EOF and normalize. Use system with capillary thermostating.
Different silanol content of Different wall charge and variations in EOF capillary batches Temperature changes Changes in viscosity and EOF
Poor Peak Area Reproducibility Sudden application of high Heating, thermal expansion of buffer, and expulsion of voltage sample Sample evaporation Instrumental limitations Increasing sample concentration and peak area System rise time significant proportion of injection time Ramp separation voltage or inject buffer plug after sample. Cap vials and/or reduce temperature of sample carousel. Increasae injection time.
Poor Peak Area Reproducibility Sample carry-over Extraneous injection Use capillary with flat, smooth injection end. Remove polyimide from end of capillary. Cannot be totally eliminated. Increase injection amount to minimize effect. Change buffer pH. Increase buffer concentration. Use additive such as cellulose or coated capillary. Optimize integration parameters. Increase sample concentration. Use peak height. Use system with capillary thermostatting.
Zero-injection caused by Extraneous injection simply dipping the capillary in the sample Sample adsorption to capillary walls Low signal-to-noise ratio Temperature changes of capillary environment Distorted peak shape (tailing) Non-eluting sample Integration errors Changes in viscosity and injection amount
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www.agilent.com/chem/ce
Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc. 2009 Printed in the USA November 1, 2009 5990-3822EN
5990-3822EN