Agilent Capillary Electrophoresis System — Technical Description

Product Note

standards available (including CEC*), and at the same time bearing in mind the future requirements of the user. This has been achieved by innovative detector design, high precision control of operational parameters, versatile automated operational features and provision of a fully operational core instrument which is equipped to perform all CE separation modes and is designed to facilitate low-cost upgrade for CE-MS interfacing. The instrument is controlled through the Agilent CE ChemStation using a self-explanatory, intuitive graphical user interface. The ChemStation control offers many advanced operational features to ensure versatility in all capillary electrophoretic separations. Every operation, from method development and routine operation to The Agilent CE system is a and applications literature and demanding research, has been versatile, fully automated system a worldwide network of CE designed to be straightforward for high performance capillary specialists. and easily mastered. With this electrophoresis (CE) separations, vision Agilent has created a comprising an Agilent CE The design objective was to precision engineered instrument instrument controlled through develop a complete easy-to-use allowing the analyst fully featured an Agilent CE ChemStation. system that provides high sensitiv- operation from routine analysis to ity diode-array detection, improved cutting-edge research. The Agilent CE system is supported quantitative analysis, full by a range of specialized capillar- automation, the capability of This product note presents the ies, buffers, method development performing all modes of capillary technical details of the full CE and total solutions kits, educational electroseparation to the highest offering from Agilent with descrip- tions and examples of relevant features and benefits.

* for abbreviations see back page

Agilent Technologies Innovating the HP Way 1 The Agilent Capillary Electrophoresis Instrument

The Agilent CE instrument (figure external gas pressure for perform- 2) contains all functional hardware ing CEC and CGE. The instrument for performing CE separations. includes features that allow The instrument houses a high- economic conversion for CE-MS voltage power supply, carousel for interfacing. Each of these is autosampling and fraction collec- described in detail in the follow- tion, injection system, on-capillary ing sections. high-sensitivity diode array detector, offline buffer replenishment station, capillary cartridge and is capable of accepting an Figure 2 The Agilent Capillary Electrophoresis instrument

2 Sample Injection from valve-induced pressure 1000 seconds, pressure range: -50 fluctuations. This results in accu- to 50 mbar). Accurate and repro- Two injection modes are provided: rate and highly reproducible ducible injection is possible from • pressure injection, and injections (figure 4) as well as volumes as low as a few µl. For • electrokinetic injection. exceptional linearity over a broad CEC separations the capillary is pressure range, even when injection packed with stationary phase and Although pressure injection is used times are very short (figure 5). therefore presents a high back- most frequently, electrokinetic When using pressure injection both pressure. In this case the external injection may be used when the the time and the applied pressure pressure source (up to capillary contains a matrix which can be selected (time range: 0 to 12 bar) can be used to inject does not allow pressure injection sample. (for example with fixed gel CGE), and for selectively injecting only one type of ion in non-EOF analy- Pressure (mbar) ses. Injection using either mode may be performed from the long end of the capillary or from the A1 post-detector (short) end of the capillary with equal precision.

Pressure Injection Sample introduction into the A2 capillary is achieved by applying pressure to the injection vial after A3 insertion of the capillary. The quantity injected is dependent on A4 the pressure and the length of time the pressure is applied. Figure 3 t0 t1 t2 t3 t4 Time [sec] Self-correcting pressure injection profile The Agilent CE system uses a unique mechanism which constantly controls the pressure and Corrected area Corrected area corrects for the rise-time effects of 35 valves and for any leakage during 0.74 0.43% 0.55% 0.39% Corrected area injection. Figure 3 shows the profile 30 Line of regression of pressure against time during 0.72 pressure injection. When applied, 25 r2 = 0.9999 the pressure to the sample vial is 0.7 increased gradually to its maximum 20 (A1). This is then reduced to a value 0.68 about half of the maximum (A2) and 15 a correction time (t2) is inserted. 0.66 10 The process is repeated (A3, A4 ) in order to apply precisely the injec- 0.64 5 tion parameters. In this way the total area under the graph is 0.62 0 calculated as the product of the Set1 Set 2 Set 3 0 200 400 600 800 1000 pressure and time settings to mbar*s provide accurate injection. Figure 4 Figure 5 The pressure system valves only Reproducibility of corrected peak area for 3 Linearity of peak corrected area for MECC operate at atmospheric pressure sets (N=6) of injections of separation of paracetamol with pressure p-hydroxyacetophenone separated by CZE injected amount (mbar*s) avoiding any detrimental effects

3 Absorbance [mAU] Capillary PVA-coated 60 leff 24.5 cm id100 µm Buffer Agilent oligonucleotide buffer 50 Inject 7s @ -10kV Voltage -25 kV 40 Temperature 30 °C Detection 260 / 8 nm 30 DNA filter

20 Figure 6 Oligonucleotide separation using the Agilent 10 Oligonucleotide Analysis Kit (part number 5063-6530) 0 5 10 15 20 25 30 35 Time [min]

Electrokinetic Injection Electrokinetic injection is performed by placing the sample vial at the capillary inlet and applying Injection Program Short-end Injection an electric field. When the anode The Agilent CE Chemstation allows Full control over sample-vial end of the capillary is inserted into the flexibility of defining a number access allows the use of the post- the sample vial the injected aliquot of functions during the injection detector capillary length as the will not truly represent the sample part of a method. Available effective separation length. Using make-up. Positively charged ions parameters for definition are: this functionality, the sample is will migrate into the capillary due • inlet and outlet vials, placed at the outlet end of the to their electrophoretic mobility injection pressure (-50 to capillary and a negative pressure is whereas neutral species will enter 50 mbar for 0 to 1000 s), applied for a set time. By reversing the capillary through the pumping • injection voltage (-30 to 30 kV the polarity of the electric field, action of the EOF. Anionic species for 0 to1000 s), and the short, 8.5-cm length of will only enter the capillary if the • wait time before or after capillary may be used for EOF is greater than their own injection. separation with the same degree of negative mobility. Inserting the precision as for normal injection cathode end into the capillary or This allows a variety of injection (figure 7). This may be used for: reversing the power supply techniques, for example: • quick purity check, polarity results in the opposite • injection of a buffer plug after • rapid screen of analytical process. The quantity loaded is the sample plug to minimize parameters, and dependent on the electroosmotic sample loss through thermal • very short analysis time. mobility, the ions electrophoretic expansion, For more information see mobility and the amount and • injections from multiple vials Agilent publication number duration of the applied voltage. If readily allowing the use of 5963-3403E. the EOF is suppressed only one transient ITP or stacking to ion species will be introduced into improve sensitivity, the capillary, depending on the • injection from multiple vials polarity of the electric field. In for online spiking for peak CGE with fixed crosslinked gels identification, and applying sufficient pressure for • washing the capillary end by sample injection may deform the dipping into water and waiting gel or extrude it from the opposite thus reducing the risk of end. Figure 6 shows the separation sample carryover. of synthetic oligonucleotides injected using electrokinetic injection. 4 Figure 7 Absorbance Short-end injection for fast analysis [mAU] Repetitive analyses of paracetamol (1), caffeine (2) and 1 2 4-hydroxyacetophenone (3) by MECC 3 10

4 Figure 8 Capillary 2 Sample carousel and lift stations 0

0.5 1 1.5 2.5 Electrode Time [min] Prepuncher

Sample Carousel

The Agilent CE system is equipped with a 48-position carousel (figure 8). The carousel can be Buffer thermostated simply by connect- replenishment ing an external water bath to the instrument (10 to 40 °C). Two vial lift stations are situated around the carousel to place vials at the Sample or buffer evaporation is sequence. In addition, a separate capillary inlet and outlet. Further, avoided by the combination of waste vial can be selected so that a third vial lift station can auto- carousel thermostating and the all flushings from preconditioning matically replenish, fill or empty use of fully-sealed vials. The vial can be collected separately to any vial offline before or during caps are pierced in the vial prevent fouling of the running the analysis. stations immediately before buffer. Random access to the vials insertion of the capillary and enables a sequence of multiple The carousel design allows electrodes or the replenishment analyses to be made from a single random access to any vial at all needle. sample with different buffer times. The carousel can be ac- systems which is mandatory for cessed during analyses because Sample vials can be exchanged at automated method development. the vials are lifted above the any time during an analysis or carousel when electrophoresis is during a sequence so that the Random access to all vials also performed. The anode and cathode actual sample capacity can be allows the carousel to operate as vials can be emptied and refilled increased. For routine analysis both buffer and sample tray and to with buffer offline so that no only two vial locations are needed function as a fraction collector valuable sample locations in the for the running buffer. For (see page 10). carousel are occupied by addi- method development more than 20 tional buffer vials, thus maximiz- different buffers can be tested in a ing the sample vial capacity.

5 Buffer Replenishment is of particular importance when Capillary Cassette and the buffer is chosen for properties Alignment Interface The Agilent CE system is equipped other than its buffering capacity with an automated buffer replen- (for example in indirect detection The Agilent CE system is equipped ishment station which can access techniques) or when the chosen with a cassette that is easily any vial before or during an buffer is not a true buffer but is opened without using tools and analysis, empty it, and refill it with acting merely as an electrolyte. In allows quick exchange of capillar- fresh buffer from a large volume addition when running a series of ies (figure 10). This allows rapid reservoir (about 500 ml). samples at high pH (> 7) the exchange of capillaries when Replenishment can be pro- pumping action of the EOF can changing methods. Further, the grammed to refill vials to a preset reduce the buffer volume of the ability to rapidly remove and level to automatically correct inlet vial — this can result in install a new capillary ensures unequal vial levels. For more laminar flow in the capillary due to minimum downtime, with the information see Agilent publica- unequal vial levels. On average entire process taking less than tion number 5963-3296E. buffer replenishment should be 1 minute. performed every three to five Increased Reproducibility analyses. The cassette contains two feet that make leveling of the capillary ends During electrophoresis, electroly- High Sample Throughput sis of the buffer solutions results easy and straightforward. The in changes in buffer composition The buffer reservoir for replenish- precision engineered capillary and pH. This can result in unstable ment has a capacity of 500 ml alignment interface ensures auto- electroosmotic flow and can also which ensures sufficient capacity alignment of the capillary detec- cause changes in selectivity. This for > 300 analyses when replenish- tion window when inserting the ing both inlet and outlet vial after cassette into the detector opening. each run (figure 9). Because capillary alignment can affect sensitivity it is important Absorbance [mAU] that the capillary is properly 1 Acetaminophen 1 4 40 2 Nicotinic acid aligned in the incident light path. 3 Acetyl salicyclic acid The dimensions of the slit are of 4 Dihydroxybenzoic acid 3 major importance in reducing 30 stray light and therefore increasing sensitivity and detector linear dynamic range (for more informa- 20 tion see Agilent publication 2 number 5963-1891E). Alignment Run # 35 interfaces are available for all 10 Run # 105 standard capillary dimensions and in addition special interfaces are Run # 200 available for use with the Agilent 0 Run # 265 Extended Light Path capillaries (see table 1). 0 Time [min] 10

Figure 9 Extended operation using buffer replenisment Capillary id 50 µm Length 56 cm Buffer 89 mM Tris-Borate, pH 8.2 Injection 100 mbars Voltage 25 kV Current 13 µA

6 The alignment interfaces are color- Capillary Thermostatting important to ensure precision, coded similarily to Agilent capillar- reproducibility and selectivity. By ies to ensure correct selection. The Agilent CE system provides actively monitoring and correcting Any capillary from a third-party stable and efficient capillary for variations in ambient vendor may be used with the thermostating through the use of temperature, the system is system, provided that it has a high-velocity recycling air flow independent of external minimum total length of 33 cm (10 m/s) which is thermostatically temperature fluctuations. The with a minimum post-detection regulated by a Peltier element. capillary temperature can be window length of 8.5 cm and a Capillary temperature is one of the regulated between 10 degrees standard outer diameter of many operational parameters below ambient (minimum 4 °C) up 365 µm. The design of the align- which can be optimized in capil- to 60 °C. This efficient high- ment interface and capillary lary electrophoresis. Temperature velocity air flow thermostating is cassette also provides the neces- affects an analyte’s electrophoretic capable of ensuring stable tem- sary protection when using packed mobility in CZE separations, peratures and effective capillary capillaries for capillary partition coefficients in MECC and cooling even when operating with electrochromatography. CEC and even the injected sample high concentration, high power volume through its affects on buffers (figure 11), for more liquid viscosity. Therefore, stable information see Agilent temperature control is extremely publication number 5963-3975E. The actual temperature of the capillary compartment is recorded throughout the analysis and can be stored with analysis data. The ability to achieve rapid temperature equilibrium allows the use of temperature gradients.

Figure 10 Minimum total length = 33 cm Capillary cassette Minimum effective length = 8.5 cm

Capillary id 75 µm Absorbance 2 Reproducibility (%RSD) Length 80.5 cm Buffer 105 mM phosphate, pH 2.5 mAU [200 nm] Peak 1 Peak 2 Peak 3 Injection 150 mbars Migration Voltage 30 kV 250 time 0.36 % 0.60 % 0.33 % Temperature 35 °C Current 130 µA 1 3 Power 4.8 W/m

Figure 11 Reproducibility of peptide map of human 0 hormone through efficient thermostating under high power 10 15 20 25 conditions Time [min]

7 High-Voltage Power Supply adapted from LC detectors, the tude. The linear dynamic range of optical design of the Agilent CE the detector allows detection of a The Agilent CE system has a detector has been specifically wide range of analyte concentra- power supply capable of applying developed for UV-visible absor- tions without resorting to sample voltages over the range from bance detection in capillary- dilution. This provides great utility -30 kV to +30 kV. The high-voltage shaped flow cells. In conjunction in the analysis of trace impurities in power supply is the heart of any with the capillary alignment inter- the presence of a major component CE instrument and as such it must faces the optical design of the and also supply a stable voltage during the detector is optimized for both for determination of chiral excess. course of an analytical run. This is standard capillaries and for With the use of the Agilent achieved by constantly monitoring Agilent Extended Light Path Extended Light Path capillaries the the applied voltage throughout the capillaries. Wavelength calibration linear dynamic range is extended analysis and actively correcting for is performed automatically on re- significantly beyond 500 mAU voltage fluctuations. The actual placing the capillary cassette and (figure 12). voltage applied during the run can can be performed manually at any be recorded and viewed during time. The response time of the Spectral Acquisition for Peak detector is sufficient to detect and analysis and stored with other Identification and Peak Purity analytical data. The power supply acquire spectra of high efficiency provides for rapid rise and decay peaks with peak widths less than Analysis times of around 1 second, which is 0.02 min. The Agilent CE ChemStation built-in of particular importance where diode array detector allows a fraction collection of peaks is High Sensitivity and Linear range of tasks. Simple spectra, isoabsorbance plots, 3-D plots, performed (see page 10). The Dynamic Range system has safety features built in and other evaluation can take place which limit maximum current and The design of the capillary align- online while analysis is in progress. power levels of 300 µA and 6 W ment interfaces reduces the For more information see Agilent respectively. The polarity of the amount of stray light reaching the publication number 5963-6977E. applied voltage is fully controlled detector allowing a linear dynamic by software providing a true range up to 500 mAU with a 75-µm The Agilent CE Chemstation, which positive or negative polarity with id capillary, with less than 1 % controls the system, enables full the outlet electrode held at ground deviation from linearity. This spectral information to be retrieved potential. The software control typically covers a concentration as well as single wavelength and enables the construction of range of three orders of magni- multiple wavelength detection. voltage, current or power gradients, and the application of voltage ramps which is of great impor- Absorbance [mAU] Absorbance [mAU] tance when analysing heat- 1200 sensitive analytes in low conduc- 14 tivity solutions. 1000 12 10 800 High Sensitivity Diode Array 8 600 Detector 6 400 The Agilent CE system is equipped 4 with a high-sensitivity diode array 200 2 detector (table 3). In CE the capillary itself acts as the detector flow 0 0 cell. Unlike many detectors in CE 5 10 15 20 25 30 35 40 9.5 10 10.5 11 11.5 12 12.5 instrumentation, which have been Time [min] Time [min] Figure 12 Analysis of trace impurities in polylysine

8 Spectral library searches and peak Absorbance [mAU] purity algorithms can greatly 250 Electropherogram facilitate method development in 200 CE. The peak purity algorithm is 150 100 especially useful for differentiating 50 peak shapes distorted by mobility differences between analyte and 2 4 6 8 10 12 14 16 18 min run buffer from those that represent actual impurities (figure 13). Spectra overlay Wavelength overlay

250 350 450 550 nm 8.12 8.22 8.32 8.42 min --> Spectral Data contains impurity <-- Agilent Extended Light Path Figure 13 Peak-purity analysis Capillaries

To further enhance sensitivity, Agilent Extended Light Path capillaries (bubble cell) can be used with the Agilent CE system. These capillaries contain an expanded diameter section (bubble) at the point of detection (figure 14). The bubble manufac- turing process is computer controlled to provide tolerances of less than 3 %. Agilent provides capillaries with a range of normal and extended light paths (table 1). This design results in corresponding increases in sensitivity over conventional straight capillaries of the same id, for more information Figure 14 see Agilent publication number Bubble cell with dye front pushed through to 5963-1889E. In the region of the highlight the extended path bubble the electrical resistance is reduced and thus the field de- zone expands radially (across the results in an unchanged zone creases. Concomitant to this is a capillary) and contracts longitudi- concentration but an increase in proportional decrease in flow nally (along the capillary), keeping optical path length corresponding velocity due to the expanded the total volume constant. This to the extension factor (figure 16). volume of the bubble. When the zone front enters the bubble its velocity decreases and the zone Capillary id Bubble Optical Alignment slit Color contracts or stacks. The sample (µm) factor pathlength (µm) dimensions (µm) code 25 5 125 120 x 80 Black 50 1 50 40 x 620 Green 3 150 145 x 145 Red Table 1 75 1 75 55 x 620 Blue Capillary internal diameters, bubble factors 2.7 202 200 x 150 Yellow and alignment interface dimensions 100 1 100 55 x 620 Blue

9 25 µm id capillaries with extended Absorbance [mAU] light paths of 125 µm can be used 50 for applications which use high power buffers e.g. high salt or SDS concentrations (see figure 15). 40 1 The dimensions of the alignment 1 50 µm 150 µm interfaces used with the bubble 30 cells are designed specifically to maintain light throughput by being extended radially, and to maintain 20 resolution by being reduced axially. This maintains sensitivity 22 10 without sacrificing resolution. A detailed description is given in Agilent publication number 0 5963-1889E. 0 2.5 5 7.5 10 12.5 15 17.5 20 Time [min] 33 Figure 15 Figure 16 Separation of protein standards in a Mechanism of zone 25 µm id capillary with bubble using 150 mM concentration using phosphate, ph 7.0 with 200 mM ammonium Agilent Extended Light sulfate Path capillaries

Capillary Gel Electrophoresis CE system, such separations in the see Agilent publication 5963- Operation bioscience field are made more 9807E. This filter allows higher accessible. High pressure is also light trans-mission levels than are High Pressure for Viscous invaluable for flushing 25 µm id available with conventional Replaceable Gels capillaries. interference filters which give light transmission of about 15 % found in The Agilent CE system can accept fixed wavelength detectors. For an external gas supply up to Spectral Filter analysis of nucleic acids the 15 bar which is internally regulated Linear polyacrylamide (LPA) is spectral filter provides high light from 2 to 12 bar. Provision of used in a variety of capillary gel higher pressure is of benefit in the electrophoresis applications and is Transmission CGE analysis of proteins and also used in some coated capillar- 70.00% oligonucleotides where highly ies. However, LPA is susceptible to viscous replaceable gels are used. degradation when exposed to high 60.00% When using a viscous polymer intensity light sources at low UV matrix, necessary for high resolu- wavelengths. In order to facilitate 50.00% tion SDS-PAGE, dsDNA and the use of LPA and LPA-coated oligonucleotide separations, capillaries without resorting to 40.00% capillaries of smaller internal changing the detector, Agilent has £ diameter ( 50 µm) require rela- developed spectral filters designed 30.00% tively high pressure (about specifically for use in nucleic acid 20.00% 8 bar) in order to replace the analysis. For more information, capillary volume within a reason- able inter-analysis time frame. With 10.00% the high-pressure option available Figure 17 as a core composite of the Agilent Spectral filter for 190 290 390 490 590 oligonucleotide Wavelength [nm] analysis

10 transmission at the detection wavelength range of 254 – 260 nm mAU Run # 73 (bandwidth 4 nm) with lower 80 transmission levels at 360 nm which is necessary for cassette 70 recognition (figure 17). The light 60 Run # 49 throughput at 260 nm is about 68 % 50 transmission. This is much larger than is found with fixed wave- 40 length detectors, which use 30 Exemplary RSD (n=6) intereference filters. In this case 20 Run # 1 for migration time: 0.35% the light throughput is around 15 % resulting in significantly higher 10 baseline noise. The spectral filter 0 is also recommended for use with 5 10 15 20 the Agilent ssDNA kit whre it significatntly extends its lifetime (figure 18). For more information Figure 18 see Agilent publication number Long term stability testing of CGE for 5963-9870E. oligonucleotide analysis using spectral filter

Fraction Collection In each case the DAD signal is Fraction Collection using used as the peak sensor, allowng Pressure Elution Although in capillary electrophore- time-independent fraction collection. In all three modes up to four sis only very small quantities of Pressure elution of fractions from peaks may be collected individu- analytes are injected, fraction the capillary can be used in most CE ally by defining their migration collection can be desirable for modes. The user must input time, or multiple peaks over a set further analysis using a different information on the time taken for time range can be collected. The CE mode, protein sequencing, migration of the buffer from the peak detector may also be used to mass spectrometry or other offline inlet to the detection window using specify collection of those peaks analytical techniques. The the elution pressure of 50 mbar which have an absorbance above a Agilent CE system offers fully (figure 18). This low pressure user-defined threshold. Because automated, software-controlled ensures minimum distortion of the sample carousel also acts as fraction collection in three other analyte zones still within the the fraction collection vial holder, different modes. Fractions may capillary and allows collection of the location of the vial for fraction be collected by pressure elution, multiple fractions from a single run. collection can be specified. Where by electrokinetic elution or from The software calculates when the multiple fractions are being CIEF analyses. The fraction peak will reach the end of the collected the Agilent CE instru- collection mode is selected from capillary and places the collection ment will collect the first fraction the Instrument menu of the vial at the outlet end at the appropri- in the specified vial and subse- graphical user interface, for more ate time. Fractions may be collected quent fractions in correspondingly information see Agilent in as little as 5 µl of solvent. Figure incremented vials. For fraction publication numbers 5963-1251E 18 shows the reinjection of a collection it is essential that the and 5963-3506E . peptide fraction collected by power supply decays in < 1 s. pressure elution from separation of a protein tryptic digest.

11 Absorbance [mAU 200 nm] Peak 4 reinjected 80 1 4 CE 6 4

2 X Y 3 2

Figure 19 0 Window for entering 20 25 30 35 40 20 30 40 pressure elution fraction collection parameters Time [min] Time [min] Figure 20 Reinjection of pressure collected fraction from peptide map single run using CZE

Fraction Collection using (figure 21). Electrokinetic elution Electrokinetic Elution of fractions is particularly appropriate when the capillary contains In this case the user is prompted a pressure resistant matrix, for for the capillary dimensions and example, fixed gels, viscous time of the peaks of interest polymers or packed solid phase particles (figure 22).

Abundance Fraction Mass peak from 1700 300 collected single fraction 1500 collection

200 1300 1100 3230.1Da 900 100 700

500 0 Figure 21 Window for entering 20 30 40 2600 3000 3400 electrokinetic elution Time [min] m/z fraction collection parameters Figure 22 MALDI-TOF analysis of single fraction collected from CGE oligonucleotide separation 12 Fraction Collection from CIEF sharply focused zones. Using the fraction collection option for CIEF the user provides information on In CIEF pressure and voltage are Figure 23 the capillary dimensions and applied simultaneously to mobilize Window for inputting CIEF migration time using pressure fraction collection parameters the separated components while (figure 23). maintaining the integrity of the

Absorbance [mAU] Absorbance [mAU]

150 7.2 6.6 5.9 5.4

125 15

100 WQQQSYLDSGIK 10 75

50 5 25

0 0

14 16 18 20 22 24 26 2 2.5 3 3.5 4 Time [min] Time [min] Figure 24 Refocussing of proteins isolated from micropreparative by CIEF

Capillary Electro- broadening from eddy diffusion. chromatography (CEC) The more uniform velocity flow profile greatly reduces this major CEC is a true fusion of CE and LC contribution to band broadening. Figure 25 and is, at present, an emerging Graphical representation of EOF generation in CEC technique in the first exciting stages of investigation. The technique may be used to separate neutral molecules, or mixtures of charged and/or neutral species. Similarly to LC, separations are achieved through partition between a mobile phase and a stationary phase. The motive force which propels neutral molecules through the capillary bed is the electroosmotic flow (EOF). The benefits of this are that the EOF has a uniform flow-velocity profile unlike LC which provides a more variable flow profile with an increased contribution to band

13 Since the flow is generated within to either or both of the buffer of application of pressure and the the capillary, longer columns reservoirs. The external gas pressure applied. All CEC function- containing very small particles (< pressure source should be oil-free ality is included in the Agilent CE 3 µm) may be used which are air or nitrogen. instrument hardware and software prohibited by pressure constraints control is provided as standard in in LC. For a comprehensive The CEC operational mode is the Agilent CE ChemStation. description of the technique see selected from the graphical user Although an external gas supply is Agilent publication number 5964- interface which changes to needed for high-pressure and CEC 5930E. Typically, in CEC separa- indicate that an external high operation (see figure 27), the tions, microbubble formation can pressure source is connected and Agilent CE instrument is fully occur as a consequence of apply- to indicate the external pressure functional for all other CE modes ing high voltage across a packed supplied (figure 26). The Agilent without this external gas pressure. capillary bed. This phenomenon CE system controls both the rate can be avoided by ubiquitous application of pressure to both inlet and outlet vials which Figure 26 eliminates bubble formation and Part of the graphical user separation breakdown, while interface for CEC operation introducing no laminar flow window because of the equally applied pressure.

The Agilent CE system is designed to internally regulate an external pressurized gas supply (£ 15 bar) to provide single or dual-vial pressurization at 2 to 12 bar. Regulated pressure can be applied

Absorbance [mAU] Column Spherisorb ODS1, 3 µm 250 x 0.1 mm 30 Mobile phase 80/20 ACNTris-HCl

Anthracene 50 mM, pH 8 Voltage 25 kV 25 Injection 5 k, 3 s Thiourea

Naphthalene Pressure 10 bar both sides 20 Temperature 20 °C Plate number 50,000 – 60,000 Nitrobenzene Biphenyl Fluoranthene Symmetry 0.95 – 0.98

15 Fluorene

1 234 5 6 7 8 Time [min] Figure 27 CEC separation of polyaromatic hydrocarbons

14 Capillary Electrophoresis/ Mass Spectrometry

The interfacing of capillary electrophoresis with mass spectrometric analysis is widely viewed as an optimum conjunction of separation power with a universal detection principle which also provides highly selective identification data. Sheath flow The Agilent CE instrument is fully settings, ...... data acquisition fitted as standard with the features settings necessary for conversion to spray chamber CE-MS operation. This provides settings, and... a versatile and fully automated front end for CE-MS separations. Designing the Agilent CE instrument such that it has built-in features Figure 28 which enable its conversion to Single point instrument control for CE-MS CE-MS capability greatly reduces the cost of opting for CE-MS analysis.

The Agilent CE-MS adapter kit • plastic UV capillary interface to to interface perfectly with the (part number G1603A) includes all allow tandem UV/MS detection Agilent 1100 Series MSD and the parts to allow interfacing of the (figure 30). Esquire-LC Ion Trap LC-MS (n). The Agilent CE to most ESI-MS instru- The capillary can be passed Agilent CE ChemStation supplied ments and includes: through the DAD for method as standard already contains the • capillary cassette designed to development or directly into the necessary software for CE-MS allow thermostating of the MS for routine operation. The operation. CE-MS option may be capillary throughout its position CE-MS probe for non-Agilent MS selected from the GUI toolbar. within the instrument (figure 29), should be purchased from the MS When using CE-MS software (part • ground cable, vendor. The Agilent CE sprayer kit number G2201AA) the Agilent • capillary safety sleeve, (part number G1607A) is designed 1100 Series MSD and the Agilent • warning labels, and 1100 Series isocratic pump used to supply the sheath liquid can be controlled directly from the GUI as shown in figure 28 (For more

Air cooling detailed information refer to Cassette publication number 5968-1328E).

to MS Detector interface

Figure 29 Inlet CE-MS cassette design and Optional outlet its position within the for regular CE mode Capillary instrument

Agilent

15 Absorbance UV Abundance MS-TIC [mAU] 50000 Angio- 300 Angiotensin I tensin II Angiotensin I 250 DRVYIHPFHL Angiotensin II MW 1296.5 DRVYIHPF 40000 200 MW 1046.2

150 30000

100 20000 50

0 10000

0 2 4 6 8 10 5 10 15 20 25 Time [min] Time [min]

Figure 30 CE-MS of peptide mixture

Agilent CE ChemStation guides the user through the CE Mode Selection programmable parameters. This The CE operational mode may be The Agilent CE ChemStation interface also allows direct access selected from the GUI. This allows comprises instrument control and to all method parameters, includ- operation in all normal CE modes data handling software, personal ing control of the vial tray, capil- and additionally includes CEC, computer and printer. The soft- lary flushing, and so on. The user CGE and CE-MS. The GUI ware runs within Microsoft® is informed about the actual status changes to indicate the opera- Windows and MS-DOS® operating of the analysis by real time indica- tional mode selected. environments. Data analysis tors. procedures range from standard integration protocols and reporting to advanced spectral library searches and peak-purity analysis. The Agilent CE ChemStation is part of a standardized platform for running CE, GC and LC instru- ments from Agilent Technologies.

Instrumental Control The Agilent CE system is operated from the Agilent CE Chemstation using a graphical user interface (figure 31). A schematic diagram of the Agilent CE instrument

Figure 31 Graphical user interface for CE

16 Method and sequence Figure 32 Method set-up window programming Method programming takes place from a single screen using easy to recognize icons (figure 32). The intuitive design of this interface reduces the time necessary for familiarization and facilitates rapid training. Table 2 lists all the method parameters that can be programmed. Individual methods and sample series can be com- bined in a sequence to fully automate any series of analysis methods and samples.

Sequences for Method Data Analysis and Reports CE Specific data reports and Development and High Sample analysis: The ChemStation provides for Throughput autointegration, integration and • corrected area (peak area / Sequences may be constructed to manual integration. time), contain any number of methods • Autointegration can be used to • apparent mobility and effective and to analyze numerous samples. set up initial integration para- mobility, (see publication This is of major benefit in method meters based on characteristic number 5968-2232E) development where numerous signals. • MW determination from SDS- methods with varying operational • Integration integrates the protein or DNA analyses, parameters may be used to electropherogram according to • pI determination for CIEF optimize a separation. Additionally the values in the integration analyses. different buffer vials may be events table. This may be used programmed within methods. This to adjust integration results by Quantitative reporting of peak allows automated method develop- changing integration events and areas as corrected peak area (area/ ment which may be performed reintegrating the signal. time) is required for accurate unattended. • Manual integration may be reporting of peaks separated by performed. CE, and is mandatory for chiral Full access is available to the analyses. Data analysis options sample tray during operation The user has a choice of 15 also include the ability to perform allowing samples to be removed different report styles or may sequence summary reports of and replaced during a sequence choose to customize the report to calibrated compounds. This is a thereby increasing the sample suit their own requirements major benefit in performing throughput available. Additionally (table 3). There are a number of precision calculations for system sequences can be updated or CE specific parameters which are suitability. modified while the sequence is included in the report and CE running. specific functions for calibration.

17 Table 2 Function Parameters/ Settings Values Programmable instrumental Power supply Polarity Positive or Negative parameters Voltage 0 – 30 kV Current 0 – 300 µA Power 0 – 6 W

Capillary conditioning Inlet /outlet vial Vial number 1– 48 Wait 0 – 1000 min Voltage 0 – 30 kV Current 0 – 300 µA Power 0 – 6 W Flushing 1 bar Pressure 0 – 50 mbar (programmable) High pressure 2 – 12 bar (includes HV) Both: Inlet/outlet 2 – 12 bar with/out simultaneous voltage

Buffer replenishment Inlet/outlet Vial number 1– 48 Time 0 – 1000 min Buffer level 0 – 1.8 cm

Injection Pressure 0 – 50 mbar 0 –1000 s Electrokinetic 0 – 30 kV 0 – 300 µA 0 – 6 W 0 – 1000 s Program Vial number 1 – 48 Voltage (0 – 30 kV) Current (0 – 300 µA) Power 0 – 6 W Pressure 0 –50 mbar Flushing 1 bar High pressure 2 –12 bar Wait 0 –1000 min

Detector Wavelength 190 – 600 nm Reference wavelength 190 – 600 nm Bandwidth 2 – 400 nm Peak width 0.01 – 1 min Response time 0.1 – 20 s Spectral data rate 0.16 – 5 spectra/s Signals 5 simultaneously Spectra All spectra All in peak Figure 33 Peak baseline and apex Typical electropherogram for OQ/PV test Threshold sample (p-hydroxyacetophenone) illustrating short run time and spectra Capillary cassette Temperature 10 degrees below ambient to 60°C (not below 4°C) mAU [192/4nm] Full capillary details entered in a table. mAU 40 Sample carousel Temperature 10 – 40 °C 70 Vials 1– 48 60 50 Timetable Voltage -30 – 30 kV 30 40 Power 0 – 6 W Current 0 – 300 µA 30 20 Inlet vial 1 – 48 20 Outlet vial 1 – 48 10 Capillary temperature 10 degrees below ambient to 60 °C 0 Lower current alarm limit 200 300 400 10 Polarity Positive or negative Wavelength [nm] Pressure 0 – 50 bar High pressure 2 – 12 bars with/out simultaneous voltage 0 Fraction collection Electrokinetic Pressure 0.5 1 1.5 2 2.5 3 3.5 4 4.5 CIEF Time [min]

18 Report Style Features

Short Quantitative text results of all integrated signals

Electropherogram + short Electropherogram plus quantitative text results

Electropherogram + detail Electropherogram, quantitative text and calibration curves

Header + short File header, quantitative text results.

GLP + short Header, sample information, instrument conditions, logbook, electropherogram, quantitative results

GLP + detail Header, sample information, instrument conditions, logbook, electropherogram, quantitative results and calibration curves

Short + spectrum Instrument conditions, electropherogram, quantitative results and peak purity plots

Detail + spectrum Header, instrument conditions, electropherogram, quantitative results and peak purity plots

Full Header, sample information, instrument conditions, electropherogram, quantitative results and peak purity plots

Library search Produces a calibrated report including library search results, peak numbers, ,migration times, librarysearch match factors, amounts, compound names

Performance Migration time, peak are, peak height, signal description, true half-height peak-width, symmetry, efficiency and resolution

Performance + library search Combines the performance and library search styles

Performance + noise Combines the performance report style with noise calculations

Performance + extended All parameters from the peak performance calculation and individual plots of each peak. Additionally - peak start and end, skew, excess, peak width, USP tailing factor, tie interval between data points, number of data points, statistical moments, plates, plates per metre, selectivity and resolution; all method relevant information (instrument, capillaries, sample and acquisition parameters and electropherogram)

HPCE mobility Quantitative text results plus apparent mobility

Table 3 Report styles available from Agilent CE ChemStation

Provision for Regulatory Compliance procedure tests the following The tests has been developed to Operation Qualification / instrumental parameters: identify and test functions of an Performance Verfication • temperature accuracy and Agilent CE system which should (OQ/PV) stability, be validated for regulatory compli- In the use of capillary electro- • voltage accuracy and stability, ance. phoresis in regulatory compliant • detector noise and drift, environments there is an increas- • detector wavelength accuracy Installation Qualification (IQ) ing demand for operational internally (holmium oxide Agilent can also supply the qualification/performance verifica- filter), appropriate test procedures and tion (OQ/PV). Using experience in • detector wavelength accuracy certification necessary for regulatory issues and in capillary externally (using test analyte), installation qualification as an aid electrophoresis, Agilent Technolo- • detector linearity, to the validation of methods and gies has developed a kit which • injector reproducibility, operation within regulatory provides instructions, methods • injector linearity, and compliant environments. and materials for performing OQ/ • replenishment system PV for the Agilent CE system. The functionality.

19 Capillary Electrophoresis Kits Capillaries • Bare fused silica capillaries of varying dimensions with precision cut ends for accurate and precise quantitation and Accessories • Agilent Extended Light Path capillaries for high sensitivity applications • PVA coated capillaries for protein analysis and suppressed EOF analyses • CEP coated capillaries for suppressed EOF analysis and replaceable gel CGE Agilent Technologies offers a range • ODS packed capillaries for CEC of capillaries, buffers, kits for Buffers • CZE buffers covering a wide pH range method development and total • MECC buffers for CE of neutral molecules • Gel buffers for DNA analysis solutions for CE analysis (table 4). Kits • Chiral method development kit Full details and ordering informa- • dsDNA analysis kit tion may be found in the Agilent • ssDNA analysis kit • Inorganic anion analysis kits CE supplies catalog. Additionally • Organic acids analysis kit numerous applications and • OQ/PV and IQ kits for regulatory compliance educational literature are available • Cation solutions kit • Forensic anion solutions kit in the form of application notes • Plating bath analysis kit and booklets, CE and GLP primers, and scientific publications Table 4 (figure 35). Kits and accessories for CE available from Agilent Technologies

Agilent Technologies Application Support Many of the scientific articles and Agilent Technpologies application application notes may be accessed support includes educational and through the Agilent home page on applications materials: the worldwide web at: • primers and CD-ROMs covering http://www.agilent.com/chem introduction, theory, appli cations and GLP requirements, • a wide range of application notes in bioscience, food and pharmaceutical analysis, and • an international network of CE specialists.

List of abbreviations

CE Capillary electrophoresis CZE Capillary zone electrophoresis MECC Micellar capillary electrochromatography The information contained in this note is CGE Capillary gel electrophoresis subject to change without notice.

CIEF Capillary isoelectric focussing ® ® CE-MS Capillary electrophoresis/mass Microsoft and MS-DOS are U.S. registered spectrometry trademarks of Microsoft Corporation. OQ/PV Operational qualification/ performance verification Copyright © 1996-2000, Agilent Technologies IQ Installation qualification All Rights Reserved. Reproduction, adaptation CEC Capillary electrochromatography or translation without prior written permission LC Liquid chromatography is prohibited, except as allowed under the GUI Graphical user interface copyright laws. SDS-PAGE Sodium dodecylsulfate- polyacrylamide gel electrophoresis Publication Number 5965-6512E MW Molecular weight LPA Linear polyacrylamide dsDNA Double-stranded deoxyribonucleic acid Agilent Technologies Innovating the HP Way 20