capillary electrophoresis guidebook

CE can be broadly described as high-efficiency separations of sample ions in a narrow bore 25-100 pm capillary tube that is filled with an electrolyte solution.. Sample solution typicall

CE can be broadly described as high-efficiency separations of sample ions in a narrow bore (25-100 pm) capillary tube that is filled with an electrolyte solution A typical schematic of an instrument setup is shown

in Fig 1

The principal components are a high-voltage power supply, a capil- lary that passes through the optical center of a detection system con- nected to a data acquisition device, a sample introduction system, and an autosampler Typically, the CE instrument is controlled by a personal computer

The capillary is first filled with the required buffer solution Sample solution (typically l-20 nL) is then introduced at the end of the capillary away from the detector (usually the anode) The capillary ends are then dipped into reservoirs containing high-voltage electrodes and the required buffer solution One electrode is connected to a cable leading to

From Methods m Molecular Bology, Vol 52 Capdary Electrophoresrs

Ed&d by K Altrla CopyrIght Humana Press Inc , Totowa, NJ

3

n High voltage supply

Fig 1 Typical instrumental setup

the high-voltage output, whereas the other (situated at the detector end of the capillary) is connected to an earthing cable Electrodes are composed

of an inert material, such as platinum Application of a voltage (for example, 10-30 kV) across the capillary causes electrophoretic and electroendosmotic movements (discussed later in this chapter) resulting

in the ionic species in the sample moving along the capillary and passing through the on-line detector A plot of detector response (usually UV absorbance) with time is generated, which is termed an electropherogram

1.1 Electrophoresis

This process is the movement of sample ions under the influence of an applied voltage The ion will move toward the appropriate electrode and pass through the detector The migration rate, or mobility, of the solute ion is governed largely by its size and number of ionic charges For instance, a smaller ion will move faster than a larger ion with the same number of charges Similarly, an ion with two charges will move faster than an ion with only one charge and similar size The ionic mobility (pE) is therefore related to the charge/mass ratio (Eq [ 11)

(1)

Fundamentals of CE Theory 5

Detector response

Fig 2 Theoretical separation of a range of catrons

tion viscosity, and r = radius of the ion

Therefore, when we separate a hypothetical mixture of ions havmg different charges and sizes, the smaller, more highly charged ions will be detected first (Fig 2)

The actual electrophoretic velocity, or speed of the solute ions, is related

to their mobilities and the magnitude of the applied voltage (Eq [2])

where v = velocity of the ion and E = applied voltage (volts/cm)

1.2 Electra-Osmotic Flow (EOF) Application of voltage across a capillary filled with electrolyte causes

a flow of solution along the capillary This flow effectively pumps solute ions along the capillary toward the detector This flow occurs because

of ionization of the acidic silanol groups on the inside of the capillary when m contact with the buffer solution At high pH, these groups are dissociated resulting in a negative charged surface To maintain electro- neutrality, cations build up near the surface When a voltage is applied, these cations migrate to the cathode (Fig 3) The water molecules sol- vating the cations also move, causing a net solution flow along the capil- lary (Fig 3) This effect could be considered an “electric pump.”

The extent of the flow is related (Eq [3]) to the charge on the capil- lary, the buffer viscosity, and dielectric constant of the buffer:

where pEOF = “EOF mobility,” IJ = viscosity, and 6 = Zeta potential (charge on capillary surface)

Ftg 3 Schematic of electroendosmotic flow

The level of EOF is highly dependent on electrolyte pH, since the &, potential is largely governed by the ionization of the acidic silanols Below pH 4, the ionization is small (8), and the EOF flow rate is there- fore not significant Above -pH 9, the silanols are fully ionized and EOF

is strong The pH dependence of EOF is shown in Fig 4 The level of EOF decreases with increased electrolyte concentration as the 6 poten- tial is reduced

The presence of EOF allows the separation and detection of both cations and anions within a single analysis, smce EOF is sufficiently strong at pH 7, and above, to sweep anions to the cathode regardless of their charge Analysis of a mixture of cations, neutral compounds, and anions would result in the electropherogram shown in Fig 5 The migra- tion times correspond to the time the individual peaks pass through the detector

The smaller anions fight more strongly against the EOF and are there- fore detected later than anions with a lower mobility Multiply charged anions will migrate more strongly against the EOF and will be detected later Therefore, pH is clearly identified as the major operating param- eter affecting the separation of ionic species, smce it governs both the solute charged state and the level of EOF

The overall migration time of a solute is therefore related to both the mobility of the solute and EOF The term apparent mobility @A) is measured from the migration time, and is a sum of both yE and pEOF:

where I= length along the capillary (cm) to detector, V = Voltage, and L = total length (cm) of the capillary

Fig 4 Varlatron of EOF with pH

Mobility values can be calculated from migration times when both ionic and neutral components are measured For instance, in the separa- tion of a five-component mixture shown in Fig 5, the mobility values for the peaks are calculated and given in Table 1

Example peak 2 =

IA = (1L / Vt) = (50 x 57 / 30,000 x 500) = 1.9 x lOA

vEOF (from peak 3) = (IL / Vt) = (50 x 57 / 30,000 x 600) = 1.58 x 10q

j~E=pA-pEOF=0.32x IO4 The negative values of PE for peaks 4 and 5 indicate that they are anions The separation of ions is the simplest form of CE and is often termed Free Solution Capillary Electrophoresrs (FSCE) The separations rely

Frg 5 Theoretical separation of a range of ionic and neutral solutes

Table 1 Calculated Mobility Values for the Peaks m Fig 5 Peak no Mlgratlon time, s PA cm2/Vs PE

When dealing with large biomolecules, such as nucleic acids, their electrophoretic mobilities may be very similar, and FSCE is often insuf- ficient for adequate resolution In this case, separations are performed in

Fundamentals of CE Theory

capillaries filled with gel solutions In Capillary Gel Electrophoresis (CGE), a sieving effect occurs as solutes of various sizes migrate through the gel filled capillary toward the detector Chapter 13 describes the excep- tional, efficient separations that can be obtained in gel filled capillaries The separation and quantitation of chiral samples are an important area

in many industries Highly efficient chiral CE separations (Chapter 14) can be obtained by the addition of chirally selective substances, such as cyclodextrins, into the electrolyte

Capillary electrochromatography (CEC), which is a hybrid between

CE and HPLC, has been developed In this technique, CE equipment is used to generate HPLC-type separations Capillaries are filled with HPLC packing material, and the application of a voltage results in the EOF pumping the mobile phase through the capillary The full details of this technology and some applications are given in Chapter 15, which is written by one of the initial developers of the technique

Sample can be introduced into the capillary by three techniques, all of which involve immersing the capillary end into the sample solution and exerting a force to inject sample into the capillary The three mecha- nisms for introduction of sample solution into the capillary are hydrody- namic, gravity, and electrokinetic All these methods are quantitative, and equations describing the volumes injected have been derived Figure

6 shows the principles of operation for the three methods

1.3.1 Pressure Differential

In this method, the sampling end of the capillary is immersed in the sample solution and a pressure difference applied (positive pressure or vacuum) The volume of sample solution injected onto the column can be calculated:

It should be noted that there is a viscosity term in Eq (5) Therefore, it

is important to match the viscosities of the samples and standards Tem- perature, therefore, has a large influence on injection volume, since vis-

Fundamentals of CE Theory

instrument suppliers that equate a sampling time to the respective vol- ume of sample (on the order of l-20 nL) introduced into the capillary

1.3.2 Gravity Injection

In this method (II), the capillary, while dipping into the sample vial,

is mechanically raised above the height of the detector electrolyte vial Typically, the sample vial may be raised 5 cm for 10 s The volume injected (12) may be calculated:

Volume = (pgAH d41YI t / 128 q 15) (6)

where AH = height difference (cm), g = gravitational constant, and p = density of the liquid

An injection volume of 6.35 nL can be calculated for a 10-s injection

at 5 cm using a capillary length of 67 cm and capillary diameter of 75 urn The following values are employed in this calculation assuming water as the buffer at 20°C density is 0.99707g/mL, viscosity is 0.8904 x 1O-2 g/cm/s, g is 980 cm/s2 The sampling variables with this technique are time and the height the sample is raised

1.3.3 Electrokinetic

In this method, the sampling end of the capillary and the high-voltage electrode are inserted into the sample solution A voltage is then applied, causing solute ions to enter the capillary by electrophoretic migration and EOF A greater number of more mobile ions enter the capillary, which can lead to sample bias effects This effect can be turned to advantage (1.3), especially when attempting to quantify trace levels of small ions The amount introduced during electrokinetic sampling is related to a variety of factors (Eq [6])

1.4 Peak EfCciency The capillary format employed in CE minimizes most sources of band broadening that occur in conventional electrophoresis or m HPLC

appllcatlon IS effectively dlsslpated through the capillary walls, which reduces convection-related band broadening encountered m conventional electrophoresrs;

ehmmates postseparation broadening effects owing to connections, and

which mmlmlzes sample dispersion during solute transport along the cap- illary, compared to the lammar flow encountered m pumped systems, such as HPLC

The major dispersive effect remaimng in CE is that of molecular dif- fusion of the solute as it passes along the capillary This diffusion IS low- est for large molecules, such as proteins, which have small diffusion coefficients Therefore, it is possible to obtain theoretical plate counts (N) of several million for biomolecules, such as nucleotldes and proteins

3 Wernberger, R , ed ( 1993) Practical Capdlary Electrophorew Acadermc, London

4 Grossman, P D and Colburn, J C., eds (1992) Capillary Electrophoreszs’ Theory and Practice Academic, London

5 Vmdevogel, J and Sandra, P., eds, (1992) Introduction to Mlcellar Electrobnetrc Chromatography Huthlg, Heidelberg

6 Camllleri, P., ed (1993) m Capillary Electrophoresls Theory and Practrce CRC, Boca Raton, FL

7 Monmg, C A and Kennedy, R T (1994) Capillary electrophoresls Anal Chem 66,28OR-3 14R

sis: operating parameter effects upon electroendosmotlc flows and electrophoretic mobllities Chromatographla, 24, 527-530

Fundamentals of CE Theory 13

9 Heiger, D N (1994) m High Performance Capdaly Electrophoresu, Hewlett Packard, Waldbronn

10 Rush, R S and Karger, B L (1990) Beckman Techmcal Bulletm TIBC 104

11 Rose, D J and Jorgenson, J W (1988) Characterisation and automation of sample mtroduction methods for capillary electrophoresis Anal Chem 60,642-648 I2 Olechno, J D , Tso, J M Y , Thayer, J , and Wamwright, A (199 1) Znt Lab May, 42-48

13 Jackson, P E and Haddad, P R (1993) Optimisatron of inJection technique m capillary electrophoresis for the determination of trace levels of anions m environ- mental samples J Chromatogr 640,48 l-487

2 High-Voltage Supply Separations are normally performed employing voltages in the region

of 5-30 kV Electrolyte ionic strengths are generally selected during method development, such that application of these voltages generates currents of 10-100 p-IA Operations with currents above this level may lead to unstable, irreproducible operating conditions On many instru- ments, it is possible to operate by applying constant voltage (most com- mon), constant current, or constant power across the capillary However, constant voltage is the most commonly employed operational mode

3 Capillaries Generally, these are composed of fused silica with typical dimensions 25-100 pm wrde and 25-100 cm long The exact length and bore of the

From Methods III Molecular B/ology, Vol 52 CapNary Electrophoresls

Edtted by K Altna Copyright Humana Press Inc , Totowa, NJ

15

Fig 1 Photograph of commercial instrument

capillary are optimized during method development The capillaries are covered with a protective layer of polyimide, which is strongly UV absorbent A section of this polyimide coating is removed to allow on-capil- lary detection Details regarding capillary treatment are given later in Section 7.4 of Chapter 3 Capillaries can also have an internal coating to alter selectivity, and these may have specific handling requirements Capillary volumes are on the order of a few microliters For example, the approximate volume of a 50-pm wide, 50-cm long capillary is 1 pL The volume can be calculated:

Standard Commercial Instrument 17

support and guarantees consistent alignment of the capillary m the opti- cal center of the detector The ends of the capillary protrude from the cartridge and dip into the autosampler vials The cartridge is manually inserted into the instrument and is then clamped into place to ensure optical alignment Cartridges are used in conjunction with cooling sys- tems The cartridge may be either filled with a coolant fluid, or cooled air may be blown through the cartridge In instruments with liquid coolant, the cartridge is filled with a fluorocarbon liquid that is maintained at the required temperature Longer lengths of capillary are coiled on a spool within the cartridge Capillaries are located and held by screws in noncartridge-based instruments Particular attention should be paid to the alignment of the capillary in the optical center of the detector (see Section 7.4 of Chapter 3)

If the capillary becomes blocked or broken, the cartridge should be fitted with a new capillary Detailed specific instructions for this proce- dure are given in the instrument manuals

5 Air Supply The majority of instruments employ a regulated air supply that is used

to perform rinses and hydrodynamic sample injection Individual instru- ments have specific requirements (detailed in the operating manuals) m terms of the type of gas required and the pressure settings

6 Temperature Control Both the sample volume injected and solute migration times are strongly dependent on temperature Therefore, it is important that a con- sistent temperature be maintained throughout an analytical sequence to ensure good migration time and peak area precision Approaches to this are instrument-specific and involve the use of Peltier cooling devices, forced air cooling, and heating ovens Temperature ranges typically employed are 20-50°C Several instruments also offer the option of cooled autosamplers, which may be of benefit when analyzing tempera- ture-sensitive samples

7 Commercial Detection Systems All commercial instruments have W absorbance detection as standard, and several now have diode array detection The majority of CE methods employ UV absorbance detection A few selected instruments also offer the possibility of fluorescence or laser-induced fluorescence detection

The UV absorbance detectors are srmilar in format to those routmely encountered in HPLC Operating wavelengths range between 190 and

760 nm, depending on the mstrument The UV lamps employed are simi- lar to those employed in HPLC Some instruments are fitted with filters, which limits the choice of wavelengths The typical portion of capillary exposed for detection purposes is 200 x 50 pm These tmy dimensions require excellent focusing of light from the UV lamp through the capil- lary This focusing is achieved through appropriate use of high-quality lens and/or fiber optics

8 Safety Given the possible hazards associated with voltages of this level, par- ticular attention is paid to ensuring adequate safety precautions by elec- trical interlocks Inadvertent attempts to access the autosampler during operation will cause shutdown of the instrument

9 Data Acquisition Device The output from a standard CE instrument is similar to that of an HPLC detector, i.e., plot of UV absorbance with time Peak integration is the preferred method of quantitatton

10 Personal Computer Controller The majority of CE systems are controlled by an external PC that externally controls all functions of the instrument The operating param- eters for each injection are preprogrammed into the PC controller For routine analysis, an identical set of parameters can be applied to a num- ber of samples

The flexibility of a PC controller is of great benefit during method development, where the instrument can be preprogrammed to evaluate several operating parameters m an unattended sequence Less elaborate systems are available that are manually programmed from a front panel

on the instrument

11 Autosampler Typical autosampler capacity ranges from 20-50 samples Sample volume requirements range from as little as 10 pL to 5 mL Numerous injections can be made from a single vial, since the injection volumes are

on the order of a few nanoliters Autosamplers can also be prepro- grammed to perform mrcropreparative fraction collection (see Chapter 9)

Standard Commercial Instrument 19

12 Consumables Autosampler vials and caps are instrument-specific and may only be available from the equipment suppliers However, suitable alternatives may be available from other sources Several reagent suppliers, such

as Fluka, and CE equipment suppliers are now developing reagent ranges specifically purified and certified for use as CE reagents Reagents avail- able include prepared buffers, SDS solutions, and electrolyte systems for performing inorganic anion and cation determinations

CI-I.~PTER 3

Kevin D Altria

1 Introduction The CE instrument is controlled by a PC The instrument settings are defined on the PC in method files Settings, such as temperature, auto- sampler vial positions, injection parameters, and rinse cycles, are defined

in the steps within the method A typical method is given below The exact settings for the parameters are determined during method devel- opment A method can be run for a single sample To analyze several samples, a sequence is created in which the method, number of samples, and injection repetitions are defined A typical method list- ing is given below Additional information concerning each process

is then described

Set temperature: Select appropriate temperature

Rinse 1: 1 -min 0 1MNaOH (or equivalent to regenerate capillary surface) Rinse 2: 2-min operating electrolyte

Injection: selected time and mode of injection

Separate: selected time and applied voltage level, detector autozero, and integrator autostart

2 Set Temperature

It is important to set an operating temperature, since both injection volume and migration time are temperature-related Typical operating ranges are 20-50°C

From Methods m Molecular Brology, Vol 52 Capillary Electrophoresls

Ed&d by K Altrla CopyrIght Humana Press Inc , Totowa, NJ

21

3 Rinse 1 (Regenerating Rinse)

In this step, the capillary is flushed with a solution to clean the capillary surface This may be performed to remove unquantified components of the previous injection, adsorbed materials, or to rehydrate surface silanols on the capillary The settings will be defined durmg the method development Typically, a 2-min rmse with O.lMNaOH may

be performed with the drops of liquid displaced from the capillary collected into an empty vial The rinse time and autosampler posi- tions for the empty vial and regenerating solution vial are entered into the method

The capillary is rinsed and filled with the run electrolyte This allows the capillary to adjust to the pH of the electrolyte A typical rinse time may be 3 min with the displaced drops from the capillary again being collected in an empty vial The rinse time and autosampler posittons for the empty vial and electrolyte vial are entered

5 Set Detector

The wavelength, absorbance range, detector rise time, and autozero details are set during this step If indirect detection is employed, then this option is selected at this stage

6 Injection

The time and mode of injectron are designated in this step along with the autosampler position of the sample vial See Chapter 1 for a descrip- tion of the various sample injection techniques available Hydrody- namic injection is routinely employed, although electrokinetic injection may be selected for sample matrix or sensitivity reasons Since differ- ent instruments have varied hydrodynamic sampling settings, it may be appropriate to define an mjection volume, in nanoliters, in addition to sampling time

7 Separate

Details regarding analysis time, autosampler positions of electrolyte vials, and the operating voltage are specified in thts step It is normal to operate with a constant voltage However, it is possible to perform analy- sis employing constant power or current

Typical Operating Procedures 23

8 Preanalysis Procedures

1 Install capillary and turn lamp on to warm up for 20 min

2 Filter the electrolyte, If appropriate, using a 0.45~pm filter or eqmvalent

3 Fill autosampler vials with electrolyte, regeneration solution, and sample

as required Ensure vials are adequately filled and that air bubbles are avoided

4 Place the vials m the positions designated in the method and/or sequence

5 Assign integrator to collect detector signal

6 Perform a test separation to ensure appropriate performance This may involve reference to system suitability criteria detailed in a specific method

4 Turn off the lamp and instrument

5 Remove vials and dispose of accordingly

10 Good Working Practices

The following points are noted, which, if adopted, will maximize the

reagents are available in specially purified form, specifically for electro- phoresis Water should be doubly distilled or HPLC grade to minimize background UV absorbance and ionic strength Prepared electrolytes are now available from suppliers (see Chapter 2, Section 12.)

2 Electrolyte preparation and storage Electrolyte should be prepared and stored in plastic bottles If pH adjustment of the electrolyte is reqmred, the

pH should be checked prior to use Alternatively, the pH-unadjusted elec- trolyte should be stored and pH adjusted immediately prior to use Glass storage bottles should be avoided since material (principally metal ions) leaches from the glass with time, causing an increase m pH A suitable shelf life may be assigned as 1 mo, unless specifically determined

3 Capillary storag+Following use, the capillary should be rinsed with dlstllled water and flushed with air To flush with air, an empty autosampler vial is placed on the autosampler and a rinse step performed from the empty vial

4 Conditionmg analyses-It is useful to conduct analyses of blanks or equtvalent at the start of a sequence to allow the system to settle

filter or simrlar to remove particulates Particulates would appear as appar- ent noise spikes on the detector baseline Samples should also be filtered if particulate matter is presented If sample filtration is performed, adequate recovery should be demonstrated during method validation

steady baseline If a filter mstrument 1s employed, the appropriate filter should be selected to allow the titter to reach the operating temperature

significantly higher than room temperature Therefore, it 1s advisable to place the samples on the instrument prior to the mltiation of the sequence This allows samples to reach a constant temperature, which is needed for consistent inlection volumes This requirement can be avoided if condi- tioning analyses are conducted (see step 4)

voltage leakage, the instrument should be regularly cleaned with cotton wool or tissue dampened with water to remove traces of electrolyte

9 Blanks-Blanks of dissolvmg solvent and/or matrix should be analyzed at the start of each sequence to allow the system to equilibrate

vent or water should be checked, especially if attempting to quantify trace levels of ions

11 Buffer depletion-Electrolysts ofthe electrolyte during electrophorests can significantly alter the concentratton and nature of the electrolyte, This effect, termed “buffer depletton,” will be exhibited by shifts m migration time and peak area Buffer replenishment, or the use of different buffer vials during a sequence, will mimmtze the effect of buffer depletion Buf- fers should be used within their buffering pH range

method to avoid cross contamination effects

11 Capillary Preparation

capillary prior to commencing use These are essential to ensure that a repeatable separation is possible when switching between capillaries

11 l Capillary Dimensions The internal and external diameter and the length (L) of the capillary

should be specified in the method (see Fig 1) A length of capillary

Typical Operating Procedures 25

Fig 1 Capillary lengths

should be measured and cut to a length slightly over that required This allows some excess for trimrning to the exact length required The length

to the detector window (1) should also be marked prior to polyimide removal Capillaries coated with a UV transparent material are avail- able from Supelco (Bellefonte, PA) and do not require polyimide removal However, these are unsuitable for use with liquid coolant-based cartridges

The fused silica capillaries generally used in CE are coated with polyimide to provide mechanical strength, because the exposed capillary

is fragile This coating strongly absorbs UV light and it is therefore nec- essary to remove the coating in the area of capillary that is to be used as the window for on-column detection The exact distance along the capil- lary to the window (I) is instrument-specific and will be given in the instrument manual

The coating can be removed by several methods The simplest method

is to place the window area of the capillary briefly (ml s) in a low-heat flame from a burner or match Avoid unnecessary time in the flame because this may cause the capillary to bend The coating will char and the burned material can be gently removed employing a tissue dampened with methanol If all the coating is not removed, further exposure to the flame is required Alternatively, the coating can be removed by gen- tly scraping the window area with a scalpel blade An eyepiece or lens should be used to ensure that all the coating has been removed from the window, Commercial capillary window burning units have recently become available that employ a wire resistor Application of

a current across the resistor causes it to heat, which is sufficient to

Fig 2 Capillary cartridge assembly

burn off the coating material Use of these, or similar units, is recom- mended because these produce a reproducible capillary window with minimal effort It is recommended that you carefully wipe the win- dow area with a methanol-dampened tissue to remove any remaining burned polyimide

The capillary window is fragile following removal of the coating and should be handled as little as,possible to avoid breakage In addition, the window area should not be touched, because grease will get onto the capillary window thereby reducing light throughput

11.3 Capillary Installation

The capillary housing is different for individual equipment manu- facturers In several instruments, the capillaries are held in cartridges whereas in others the capillary is held by retaining devices The car- tridges often incorporate a spool around which the capillary is spooled (Fig 2) In all cases it is essential to align the window area of the capillary in the optical centre of the capillary housing device Exact details of this aligning process will be given in the instrument manual

Typical Operating Procedures 27

The alignment should be checked wherever possible using an eye- piece or lens

The ends of the capillary should be trimmed to the required length using a cutting stone to ensure a neat edge The cutting stone should

be used to lightly score the capillary and cut through the polyamide protection Use of excessive force will result in the capillary end being crushed If the capillary end is not cut correctly, peak tailing and poor precision may be obtained because sample solution can become irreproducibly trapped in the jagged end of the capillary Depending on the instrument, this trimming may be needed prior to window alignment

Careful measurements of the capillary length are required to ensure correct alignment of the capillary during installation Use of too long a length can result m the capillary becoming broken during operation If the capillary is too short, it will not dip into the electrolyte reservoirs or sample solution

The majority of capillaries employed in CE are composed of uncoated fused silica Fused silica is treated in a high-temperature oven and the surface therefore has no residual silanols To rehydrate this surface, a rinse with a high pH solution is required (I) Following this rinse the surface silanols are restored and a consistent electroendosomotic flow (EOF) is possible It is recommended that a rinse with O.lMNaOH for at least 20 min should be used to regenerate the capillary prior to its initial use The capillary should then be filled with the method-specific electro- lyte A voltage should be applied across the capillary for 20-30 min prior

to injection of the first sample This procedure is to allow time for the capillary to stabilize and to achieve a consistent EOF level This stabili- zation period could alternatively be accomplished by incorporation of appropriate blank injections at the beginning of a sequence Use of rehydration steps for capillary coatmgs should be avoided unless pre- scribed in the instructions supplied with the capillary

Once the capillary is regenerated, it is not necessary to rehydrate it with an extended NaOH rinse before each subsequent use, providing that

it is stored correctly The capillary should be labeled and reserved for use solely for a selected electrolyte Failure to do this may result in severe problems with irreproducible separations

11.5 Capillary Storage

Prior to storage, capillaries should be flushed with water and then air

to remove electrolyte from the capillary Storage filled with air prevents formation of a gel layer inside the capillary that can form if the capillary

is stored filled with solution (2) If the capillaries are stored filled with electrolyte, blockages can form as the electrolyte crystallizes out

References

1 Coufal, P., Stulik, K., Claessens, H A , and Cramers, C A (1994) The magmtude and reproducibllrty of the electroosmotic flow in sihca capillary tubes JHRCC 17, 325-334

2 Schwer, C and Kenndler, E (1991) Electrophoresls m fused-silica capillaries: the influence of orgamc solvents on the electroosmotlc velocity and the zeta potential Anal Chem 63,180 l-1807

CT~APTER 4

Kevin D AZtria

1 Introduction This chapter provides some possible starting points for method devel- opment and further optimization A later subsection in this chapter pre- sents some starting points and guidelines for method development of chiral separations A more extensive treatment of chiral separations is given in Chapter 14 A detailed background to the parameters affecting MECC separations is given in Chapter 12

Before proceeding with any CE experimental work, the following physicochemical information on the sample should be obtained if available

1 The aqueous solubihty of compound over a pH range;

2 Suitable dissolving solvent, preferably water; dtlute acid or base (> 10 rr&I),

or water with the mimmum level of organic solvent required to solubke the compound;

3 pK, data;

4 UV spectral information

Many of the variables used to adjust selectivity are similar to those employed in HPLC method development However, additional variables are also exploited (Table 1) The majority of these parameters must be optimized during method development

Resolution and analysis time are dependent on several factors (I) Therefore, it is necessary to follow a logical method development path, such as that given below:

1 Select electrolyte;

2 Select capillary and dimensions;

From Methods m Molecular Bology, Vol 52 Capillary Electrophoresls

Edlted by K Altrla Copyright Humana Press Inc , Totowa, NJ

29

Table 1 Variables AvaIlable to Develop/Optlmlze Separation Variable Typical range Effect of increasmg vanable

Various

1 5-11.5

1 O-200 n-&l l-30% v/v l-7M l-20 M l-50 mM l-100 n04 Various 5-200 mM I-20 mA4 I-20 s

Reduced analysts time and some loss m resolution Reduced analysis time and some loss m resolution Increased analysis time and gam m resolution Increased current, better sensltlvlty, and possible efficiency reduction

Change of EOF and selectlvlty Increased EOF, Increased lomzatlon of acids, and reduced lomzatlon of bases

Increased retention, lower EOF, and selectivity changes

Increased solublllzatlon, reduced EOF (except acetomtnle), and alteration m selectivity Increased solublllzatlon of hydrophobic solutes and Increased migration times

Can reduce or increase solute charge, and can alter selectivity

Reduced surface charge and reduced peak tailing Increased vlscoslty, reduced EOF, and increased solute migration if complexation occurs Reduce EOF and longer migration times Increased current, EOF and solute lomzatlon, and

reduced peak tailing Reversal of EOF dfrectlon and MECC condltlons Improved signal, some loss of resolution, and loss of peak symmetry

3 Optimize temperature;

4 Optimize wavelength selection;

5 Optimize sample concentration and compositlon;

6 Determine voltage/current;

7 Determme rinse cycle selection,

8 Optimize injection selectlon;

9 Optlmlze precision (see Chapter 6);

10 Optlmlze sensitivity (see Chapter 7)

2 Select Electrolyte Choice

There are three main separation mode options available in CE, and selec- tion largely depends on the charge of the test solute in solution (Fig 1)

Yes

0

High pH Coated -ve voltage

No Yes

High pH MECC Uncoated +ve voltage

Low pH MECC Coated -ve voltage

Fig 1 Method development options flowchart

Option 1: Low-pH free solution CE (FSCE): Cationic solutes separate by

virtue of their differing mobilities

Option 2: High-pH FSCE: Anionic species separate by virtue of their dif-

fering mobilities

Option 3: Micellar electrokinetic capillary chromatography: Solutes chro-

matographically interact with a migrating micellar phase; this mechanism allows separation of both charged and neutral species

Capillaries can be internally coated to alter selectivity or to reduce EOF (see Section 3.) These coatings can be either permanent (chemi- cally bonded) or temporary (use of electrolyte additives) Permanent coatings are usually achieved by derivatization of the capillary wall silanols followed by a covalent binding or material, such as polyacryla- mide Electrolyte additives, such as polyvinyl alcohol or ethylene gylcol, are used to reduce or eliminate EOF Surfactant additives, such as tetradecyltrimethylammonium bromide, can be employed as additives to reverse the EOF direction (this is used especially in the analysis of small anions see Section 1 of Chapter 20)

If p& data are available on the test compound, an informed selection

of electrolyte can be made If the compound is basic, then option 1 would

be preferred If the compound is acidic, option 2 would be most appro-

priate Option 3 would be applicable in all cases However, if no p& data are available, it may be appropriate to perform preliminary method scout- ing experiments to cover all of the above options The test solution should

be prepared in water or in water with the minimal amount of organic solvent

to solubilize the analyte (see Section 6.) Sample concentration should

be adequate to produce a measurable response; typically an initial sample concentration of -0.1 mg/mL may be appropriate The sample solution must be soluble in the electrolyte, or it will precipitate out in the col- umn, resulting in no peaks being detected The test solution could then

be analyzed under the following three starting conditions:

Electrolyte A: 25 mMNaH,PO, adjusted to pH 2.5 with cont H3P04 Electrolyte B: 25 mM borax

Electrolyte C 20 mM Na2HP04, 10 mM borax, with 50 mM SDS

Typically, set a run-time of 30 mm, and apply +25 kV across a 57 cm

x 50 pm capillary for each separation Set the detector to 200 nm or an appropriate UV absorbance maxima (if known) for the selected com- pound If no peak is obtained using electrolyte A, then the compound is uncharged or only marginally ionized at low pH, which effectively elimi- nates option 1

When using electrolyte B, the electroendosmotic flow (EOF) in the system will result in a negative peak at about 2-4 min This position is similar to the dead volume marker in HPLC If the sample comigrates with the EOF, then it is neutral under these conditions, which would effectively eliminate option 2

Using option 3, a peak will be obtained between 3 and 30 mm It may then be necessary to alter conditions to achieve the required selectivity

For the method scouting experiments, use typical method setup as given below:

Step I: Rinse cycle 1: O.lMNaOH (2 mm)

Step II: Rinse cycle 2: electrolyte A, B, or C (2 mm)

Step III: Set detector: select desired iL

Step IV: Sampling 1 s hydrodynamic

Step V: Operating voltage: + 25 kV

Run time: 30 mm

Capillary dimensions: 50 urn x 57 cm (50 cm to detector)

Development / Optimizatton 33

Table 2 Commonly Used Electrolytes Electrolyte

Phosphate 2 12 pK,l

7.21 pK,2

12 32 pK,3 3.06 pK,l

5 A0 pK,2

4 75

6 15 6.80

2.1 Free Solution CE

A list of common buffers and pH ranges used in FSCE is given in Table 2 The inorganic electrolytes, such as phosphate and borate, are commonly used at concentrations in the area of 10-50 mM Higher concentrations are not generally possible because of internal heating problems within the capillary, but can improve peak shape The blologi- cal or “Goods buffers,” such as CAPS, TRIS (Tris-[hydroxymethyl]- aminomethane), and so on, can be used at higher concentrations because

of their low conductivities, but problems can occur owing to their high

UV absorbances compared to inorganic electrolytes, such as phosphate

or borate

2.2 Low-pH FSCE (Option 1) The principal variables that can be employed to alter separation selec- tivity at low pH for the separation of cationic solutes are listed below:

1 Electrolyte pH: This IS the most useful parameter to vary since it can be

used to alter the charged nature of the solute To ensure full protonation of the compound, a pH of 1 or more units below its p& should be selected

2 Cyclodextrm concentratton: These carbohydrate addmves are available m

a range of native and derivatized forms and may differentially complex wtth the migrating solute, thereby increasing its migration time and sepa- ration selectivity Typically l-1 00 rnA4 hydroxy- propyl+-cyclodextrm (2)

or similar are utilized

3 Organic solvent Solute pK,lpKbs are altered by the addition of orgamc solvents Typically acetonitnle, methanol, or isopropanol at levels of l-30% v/v are employed often to improve the solubility of the solute (3,4)

4 Ion-pair reagents: These additives are used to alter the net charge of the solute Typically, l-40 mA4 sodium heptanesulfomc acid (2,5,6) or similar ton-pair reagent are added to the electrolyte

5 Electrolyte nature and concentration: Higher ionic strength buffers improve peak shape, but generally necessitate a reduced applied voltage level since the current increases with buffer concentration It 1s recommended that the voltage be reduced sufficiently to maintain current levels below 100

PA The use of Goods buffers (7) at high concentrations can be advanta- geous, since these generate relatively low currents compared to inorganic electrolytes, such as phosphate or borate

The choice of electrolyte at a particular pH also affects both the selec- tivity and EOF level For instance, the level of EOF decreases acetate > phosphate > citrate > borate (8) Also, the level of current generated by a specific electrolyte can be reduced by switching to an electrolyte counterton having a smaller ionic radius, 1.e , using lithium acetate instead

of sodium acetate (lithium dodecyl sulfate is commercially available, which can be used at higher concentrations m MECC than SDS)

Higher ionic strengths generally lead to improved peak efficiencies (Fig 2), because sample “stacking” is improved Figure 2 shows the analy- sts of a peptide mixture using various electrolyte concentrattons

6 Zwitterionic additives: Compounds, such as betame, gylcine, and taurine, can be added to the electrolyte to reduce tailing effects Given their zwtt- teriomc nature they can be used at lOO+ rnJ4 concentrations with no significant impact on overall current (9)

EOF can be reversed by addition of catronic surfactants (Z&I I) or polybrene (12) These additives form a double layer at the capillary wall, resulting in a net positive charge Application of a voltage therefore results

in a reversal of the conventional EOF direction Therefore, a negative applied potential is employed to cause a flow m the direction of the detector

8 Amine modifier: Excessive peak tailmg for highly basic solutes, such as

Fig 2 Effect of electrolyte concentration on resolution

to reduce this interaction by removing the active sites

This separation option is useful for the separation of anionic solutes The variables are similar to those considered for low-pH FSCE To ensure full ionization of the compound a pH, 1 or more units above its pK,, should be selected EOF is more important at higher pH values and

is decreased by decreases in pH, increases in organic solvent content (except acetonitrile), and the addition of cyclodextrins Since the solutes

are now anionic, the nature of the ion-pair reagent should be changed to

2.4 MECC (Option 3)

This option is useful for the separation of charged and neutral species (a more detailed background to MECC [also known as micellar electro-

in optimization of MECC separation have been summarized (14,1.5) The selectivity variables and effects are identical to high-pH FSCE How- ever, there are also several additional parameters that greatly affect both selectivity and migration times:

1 Surfactant type: Altering the nature of the surfactant greatly affects the chromatographic mteractlons with the mlcelle The prmclpal alternative to

solute is anionic under the separation conditions, it may be appropriate to employ a catlomc surfactant, such as cetyltrimethylammontum bromide, and

to apply a negative potential (18) Noniomc surfactants, such as Brig-35, can also be added to SDS-based MECC electrolytes to alter selectivity (f 9)

2 Surfactant concentration: Increased concentration results m a higher number of micelles, and therefore, the solute 1s more retained, result- ing in a longer migration time There is an optimal surfactant concen- tration for each separation, and a range should be exammed during method development

3 Cyclodextrins: These are useful as selectivity manipulators m MECC, especially when separating hydrophobic compounds

4 Urea: This additive can be employed to increase solubihzation of hydro- phobic compounds if they are poorly retained in the mlcelle (20)

and/or decrease SDS concentration

modifier or additive

centration

and increase SDS concentration

Given that optimization can be achieved through adjustment of an assortment of variables, the appropriate use of experimental design pro- cedures can significantly reduce the number of experiments required A detailed section on experimental design is given in Chapter 20 Overall,

Development/Optimization 37

the electrolyte should be chosen that gives low UV absorbance at the detection wavelength, good buffering capacity at the pH required, and sufficiently low conductance to give stable operating currents

3 Select Capillary and Dimensions Capillaries are almost exclusively composed of fused silica material, which is relatively cheap and readily available for a number of capillary suppliers, such as Polymicro (Phoenix, AZ), SGE (Ringwood, Victoria, Australia), and Supelco (Bellefonte, PA) Generally, these capillaries are not internally coated and have bare internal walls However, there are a number of examples where capillaries are internally coated to modify the level of EOF and to alter selectivity This area has recently been reviewed (21) Coatings are often with polymers, such as cellulose or dextran (22), which effectively suppress EOF and can reduce sample adsorptton onto the capillary wall Alternatively, the capillaries can be internally coated with such substances as polyethylenimine (23), which can induce an effective positive charge on the capillary wall, resulting in a reversal of electroendosmotic flow direction Alternatively, the capillary walls may

be coated with long-chain (C6-Cl8) hydrocarbons (24,25) This is achieved following reaction of the capillary wall with appropriate silanes

A detailed procedure for preparation of bonded phase capillaries has been published (26) Alternatively, capillaries internally coated with bonded polyacrylamide are also employed Coated capillaries are available from both a number of CE instrument suppliers and capillary manufacturers The coated capillaries are generally supplied with handling/rinsing instructions and often with recommended electrolytes for particular applications However, it is stressed that the majority of CE applications are performed in bare fused silica capillaries The choice of capillary bore and length largely governs the speed and sensitivity of the method

3.1 Capillary Length Use of a longer capillary increases migration times for two reasons First, the length of capillary to the detector area is increased, and in addi- tion, the voltage gradient (V/cm) is decreased moving to a longer capil- lary while maintaining the same applied voltage Therefore, a doubling

in capillary length dramatically increases migration time, but also gives improved resolution Figure 3 shows use of the same separation per- formed on both a 27- (Fig 3A) and a 57-cm capillary (Fig 3B) Limita-

Fig 3 Effect of capillary length reduction on resolution (A) 57 cm; (B) 27 cm

tions to the minimum capillary length possible are specific to individual commercial instruments If attempting to employ a relatively high electro- lyte concentration, it may be advisable to apply a low voltage (i.e., l-5 kV) across a short capillary to avoid problems of excessive current generation

Development /Optimization 39

3.2 Capillary Bore The choice of capillary internal diameter largely governs the sensittv- ity of the method If sensitivity is not an issue, then it is advisable to employ a 50 pm capillary to avoid any problems with excessive current generation For maximum sensitivity, the bore may be extended to 100

pm if necessary, but at the expense of a reduced voltage and/or electro- lyte concentration since internal heating problems increase with capil- lary diameter

4 Optimize Temperature Temperature plays an important part in many separations, because both solute mobility and the level of EOF are temperature-related The major- ity of commercial instruments have capillary thermostatting facilities, and typical operating ranges are on the order of 25-6O”C Temperature can have a marked effect on selectivity in MECC, where increased parti- tioning occurs at higher temperatures On-column chelation, for example, interactions between borate ions and sugars, may also be increased at higher temperatures (2 7)

5 Optimize Wavelength Selection The maJority of electrolyte systems employed in CE have only limited

U activity, and it is therefore possible to operate in the 190-220 nm UV region, which is generally inappropriate in HPLC Many compounds have significantly greater UV activity in this region, and these UV wave- lengths are widely employed in CE Alternatively, it may be appropriate

to employ indirect detection

6 Optimize Sample Concentration and Composition

This parameter should also be optimized during method development Excessive sample concentration can lead to severely distorted peaks Generally, peak shape becomes more triangular as sample concentration

is increased excessively When attempting to determine trace impurities, these high concentrations may not necessarily be avoided However, if possible, use of lower concentrations will result in more symmetrical peaks, which will result in improved resolutions In addition, excessive sample concentrations may lead to distorted peaks if the sample has only

a limited solubility in the run buffer Operation at high temperatures can reduce on-column solubility problems, but will alter selectivity

The efficiency and performance of a CE separation can also be greatly affected by the presence of undesired sample components, such as high levels of salt or organic solvent The most appropriate sample solutions are water or a 1: 10 dilution of the run buffer If the ionic strength of the sample solution is lower than the run buffer, a focusing of the sample solution, known as “stacking,” occurs during the initial sec- onds of the separation Stacking improves peak efficiency and can greatly improve resolution A full treatment of sampling stacking and its limitations is given in Chapter 16

Samples having a high ionic strength present the most difficulties m

CE In these circumstances, the stacking process works against the tech- nique, and results m band broadening and loss of resolution This can result in severe difficulties, since many biological samples analyzed may contain high levels of salt To minimize this disruption, the use of short mjection times is recommended If sensitivity permits, dilution of the sample will also reduce the effect If these approaches are inappropriate, then a sample pretreatment, such as solid-phase extraction, may be required to remove ionic interferences

When attemptmg longer injection ttmes of high-ionic-strength samples, run failures may occur These events are caused by boiling of the sample solution during the initial seconds of separation, because most

of the heat initially generated would be in the sample zone area If this occurs, it IS necessary to reduce the sampling time and reinject

High levels of organic solvent in the sample solution can also have an undesired impact on the quality of a separation (28) Again, the best strat- egy is to minimize the sample injection time Poor water solubility may require high levels of organic solvents Therefore, higher sample con- centrations may be appropriate Alternatively, it may be appropriate to dissolve the sample m dilute acid or base (29) if possible Sample stabil- ity in the drssolving solvent will need to be evaluated

If excessive sampling times are attempted with samples containing high organic solvents, run failure can occur This failure is owing to out-gassing of dissolved gases in the solvent

Ultimately, sample solvent composition is dependent on the solubility

of the sample When performing quantitative analysis, it is important to match the viscosities of all samples with each other and the standards, because the volume injected is related directly to the sample viscosity If

c!ONDn-XON.9

CnpUhr&Ocm(Ld)x63Bcm~t~

Temperature= 26’C BuITer 0 03M SBS and

in 66% Waler/16% Me011 PEAK IDENTIF-ICATION l- NIACINAMIDE

Fig 4 Influence of voltage on resolution (From ref Z)

it is impossible to match viscosities, use of an internal standard will com- pensate for this problem

7 Determine Voltage/Current This factor (I) largely affects the speed and quality of a separation Application of a high voltage reduces analysis time, but may lead to sig- nificant losses of resolution and peak efficiencies if excessive heating occurs within the capillary The choice of operating voltage should be optimized in conJunction with the choice of electrolyte concentration, capillary dimensions, and temperature to produce an acceptable level of current Figure 4 shows that resolution is improved at lower voltages, but

at the expense of increased analysis time

Many instruments may be operated in constant current mode, which may

be an advantage if temperature fluctuates withm a separation sequence How- ever, it is advisable to operate generally in constant voltage mode, since slight mterday variations in electrolyte composition will have an impact

on the conducttvity, which may alter the run current significantly

It is important to maintain a consistent EOF run-to-run since any varia- tion results in poor migration time precision (30) Sample components, such as proteins, can become adsorbed onto the capillary surface and change the effective charge on the wall, resulting in a reduction m EOF The adsorped material can also chromatographically interact with the solute and cause tailing To prevent difficulties owing to adsorption and

to ensure a consistent EOF, the capillary 1s flushed between injections with a dilute sodium hydroxide solution that effectively strips the top surface of the capillary wall Typically, a 1-min rinse with 0 IM NaOH

is sufficient The capillary is then flushed with the buffer prior to injec- tion Other rinsing regimens may involve use of a dilute acid solution or

a strong buffer solution For example, if the run buffer is 20 mA4 phos- phate, it may be appropriate to rinse with 100 mk! phosphate It is not obvi- ous whether a rinse step should be included in a particular method Generally,

it is better to include one to prevent potential problems If the sample is an uncomplicated matrix, then it may be possible to avoid a rinse step

During method development, the rinse step should be optimized to give a good migration time precision Having established the optimum buffer conditions, ten replicate analyses should be conducted of a typical sample solution using the selected rinse step(s) between injections If the migration time precision is poor, it may be necessary to extend the rinse times or include additional steps Alternatively, if good migration time precision is obtained, the possibility of reducing the time or number

of rinse steps should be evaluated

During robustness testing, the time and concentration of rinse solu- tions should be varied to assess their impact on the performance of the method Following this robustness, testing limits can be put on the rinse time(s) and rinse solution concentrations

It is also important to allow a capillary to become adjusted to new buffer conditions Therefore, it may be appropriate to employ an extended rinse (i.e., 5 min) with buffer at the start of a sequence or change in buffers during method development