Capillary Electrophoresis Guidebook: Principles, Operation, and Applications (Methods in Molecular Biology Vol 52)

CHAPTER1

Fundamentals of Capillary Electrophoresis

Theory

Kevin D. Altria 1. Introduction This section will describe the fundamental theory, equations, and definitions necessary to comprehend the concepts involved in capillary electrophoresis (CE). This is not an exhaustive treatment, but is considered sufficient to comprehend and appreciate the principles of CE. More detailed theoretical background can be obtained from a number of reference books (I-6). Developments in the field of CE are reviewed in detail annually in the journal Analytical Chemistry. For example, the 80 1 papers published in 1992-l 993 were recently reviewed (7). 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 capillary that passes through the optical center of a detection system connected 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

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4 n

High voltage supply

Empty vial

0

Fig. 1. Typical instrumental setup.

the high-voltage output, whereasthe other (situatedat the detector end of the capillary) is connectedto an earthing cable. Electrodesare composed of an inert material, such as platinum. Application of a voltage (for example, 10-30 kV) across the capillary causes electrophoretic and electroendosmoticmovements(discussedlater in this chapter) resulting in the ionic speciesin the samplemoving along the capillary and passing through the on-line detector. A plot of detector response(usually UV absorbance)with time is generated,which is termedan electropherogram. 1.1. Electrophoresis

This processis the movementof sampleions under the influence of an applied voltage. The ion will move toward the appropriateelectrodeand passthrough 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 chargeswill move faster than an ion with only one charge and similar size. The ionic mobility (pE) is therefore related to the charge/massratio (Eq. [ 11). (1)

Fundamentals

of CE Theory

5

Detector response

Fig. 2. Theoretical separation of a range of catrons. where PE = electrophoretic mobility, CJ= number of charges, IJ = solution 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 speedof the solute ions, is related to their mobilities and the magnitude of the applied voltage (Eq. [2]). v=pEE

(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 electroneutrality, cations build up near the surface. When a voltage is applied, these cations migrate to the cathode (Fig. 3). The water molecules solvating the cations also move, causing a net solution flow along the capillary (Fig. 3). This effect could be considered an “electric pump.” The extent of the flow is related (Eq. [3]) to the charge on the capillary, the buffer viscosity, and dielectric constant of the buffer: pEOF=(&&/q)

(3)

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

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Ftg. 3. Schematicof electroendosmoticflow. 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 therefore 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 potential 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 migration times correspond to the time the individual peaks pass through the detector. The smaller anions fight more strongly against the EOF and are therefore 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 parameter 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: PA = pE + JJEOF= (ZL/ tV) (4) where I= length along the capillary (cm) to detector, V = Voltage, and L = total length (cm) of the capillary.

Fundamentals

of CE Theory

7

15

1

05

0 3

4

5

6

I

9

PH

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 separation 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

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8

Frg. 5 Theoretical

separation

Calculated Mobility

of a range of ionic and neutral solutes. Table 1 Values for the Peaks m Fig. 5

Peak no

Mlgratlon time, s

PA cm2/Vs

1 2 3 4 5

400 500 600 750 900

2.38 x lo” 190x1@ 1.58 x 1W’ 1.27 x lo” 1.06 x lti

PE 080x lOA 0 32 x lOA 0 -031 x lOA -052 x 10“

I = 50 cm, L = 57 cm, and V = 30,000 V

principally on the pH-controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. In FSCE, all neutral compounds are swept, unresolved, through the detector together (Fig. 5). Separation of neutrals is generally achieved by incorporation of anionic surfactant, at sufficient concentration to form micelles. These anionic micelles migrate against the EOF and can chromatographically interact with neutral solutes. Solutes having a large interaction will migrate later than those having little or no interaction. Use of micellar solutions is known as micellar electrokinetic capillary chromatography (also called micellar electrokinetic chromatography) and is covered in depth in Chapter 12, which is coauthored by the originator of the technique. When dealing with large biomolecules, such as nucleic acids, their electrophoretic mobilities may be very similar, and FSCE is often insufficient 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 exceptional, 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. 1.3. Sample

Introduction

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 mechanisms for introduction of sample solution into the capillary are hydrodynamic, 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: Volume = AP d411t / 128 q L (5) where AP = pressure difference (mbar), q = buffer viscosity, L = total capillary length, and d = capillary diameter (pm). Table 2 gives injection volumes (9) for l-s injections using 65-cm capillaries of varying bore, TJ= 1, and various AP values. These volumes generally correspond to sample plug lengths of 10 rr&I), or water with the mimmum level of organic solvent required to solubke the compound; 3. pK, data;

4. UV spectralinformation. 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. Selectelectrolyte; 2. Select capillary and dimensions; From

Methods m Molecular Bology, Vol 52 Capillary Electrophoresls Edlted by K Altrla Copyright Humana Press Inc , Totowa, NJ

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30

Al trta Table 1 Variables AvaIlable to Develop/Optlmlze

Variable

Typical range

Voltage Current Capillary length Capillary bore

5-30 kV 5-250 PA 20-100 cm 25-100 pm

Capillary coating PH

Various 1 5-11.5

Surfactant m MECC

1O-200 n-&l

Organic solvent

l-30% v/v

Urea

l-7M

Ion-pair reagent

l-20 M

Amme modifiers Cyclodextrms

l-50 mM l-100 n04

Vlscoslty Electrolyte cone

Various 5-200 mM

Catiomc surfactant Injection time

I-20 mA4 I-20 s

3. 4. 5. 6. 7. 8. 9. 10.

Separation

Effect of increasmg vanable 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

Optimize temperature; Optimize wavelength selection; Optimize sample concentration and compositlon; Determine voltage/current; Determme rinse cycle selection, Optimize injection selectlon; Optlmlze precision (see Chapter 6); Optlmlze sensitivity (see Chapter 7).

2. Select Electrolyte

Choice

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

Development /Optimization

YES

YES

0

u

No

0 u Low pH Uncoated +ve voltage

NO

Yes 0 Low pH Coated +ve voltage

0

MECC

FREE SOLUTION CE

No

NO

0

High pH Uncoated +ve voltage

Yes

0

High pH MECC Uncoated +ve voltage

Yes

0 Low pH MECC Coated -ve voltage

High pH Coated -ve voltage

Fig. 1. Method development options flowchart. Option 1: Low-pH free solution CE (FSCE): Cationic solutes separateby virtue of their differing mobilities. Option 2: High-pH FSCE: Anionic speciesseparateby virtue of their differing mobilities. Option 3: Micellar electrokinetic capillary chromatography: Soluteschromatographically 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 (chemically 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 polyacrylamide. Electrolyte additives, suchaspolyvinyl alcohol or ethylene gylcol, are used to reduce or eliminate EOF. Surfactant additives, such as tetradecyltrimethylammoniumbromide, can be employed as additives to reversethe EOF direction (this is usedespecially 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 compoundis basic,then option 1 would be preferred. If the compound is acidic, option 2 would be most appro-

Al tria

32

priate. Option 3 would be applicable in all cases. However, if no p& data are available, it may be appropriate to perform preliminary method scouting 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 column, 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 compound. If no peak is obtained using electrolyte A, then the compound is uncharged or only marginally ionized at low pH, which effectively eliminates 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 Citrate Acetate MES PIPES ACES MOPS0 MOPS HEPES Trls Borate CHES CAPS

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 6 90 6 90 7 20 7 55 8 30 9 24 9 50 1040

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 blological 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 selectivity 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

usedto alter the chargednatureof the solute.To ensurefull protonation of the compound, a pH of 1 or more units below its p& should be selected.

34

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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 separation selectivity. Typically l-1 00 rnA4hydroxy- 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 mA4sodium 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 1srecommended that the voltage be reduced sufficiently to maintain current levels below 100 PA. The use of Goods buffers (7) at high concentrations can be advantageous, 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 selectivity 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 analysts 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 zwttteriomc nature they can be used at lOO+ rnJ4 concentrations with no significant impact on overall current (9). 7. Flow reversal by hydrodynamic coating of the capillary-the direction of 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 peptides, can occur when they interact with the acidic silanols on the cap-

35

Development/Optimization

O.lOO M

0.060 M

4

II

Time

10 ( Minutes

la

14

)

Fig. 2. Effect of electrolyte concentration on resolution. illary surface. Additives, such as diaminopropane (13), can be employed to reduce this interaction by removing the active sites. 2.3. High-pH

FSCE

(Option

2)

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

36

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are now anionic, the nature of the ion-pair reagent should be changed to typically l-10 mM tetraethylamtnonium bromide (TEAB).

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 electrokinetic chromatography] is given in Chapter 12). The options available in optimization of MECC separation have been summarized (14,1.5). The selectivity variables and effects are identical to high-pH FSCE. However, 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 SDS 1sto employ bile salt surfactants (16,17), such as sodium cholate. If the 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 1smore retained, resulting in a longer migration time. There is an optimal surfactant concentration 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 hydrophobic compounds if they are poorly retained in the mlcelle (20). If the separation 1sgood, but analysis times are too long, increase pH and/or decrease SDS concentration. If the migration times are long with poor resolution, use an organic modifier or additive. If the migration times are short with poor resolution, increase SDS concentration. If the migration times are short with moderate resolution, decrease pH, and increase SDS concentration. l

l

l

l

Given that optimization can be achieved through adjustment of an assortment of variables, the appropriate use of experimental design procedures 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 stressedthat 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 addition, the voltage gradient (V/cm) is decreased moving to a longer capillary 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 performed on both a 27- (Fig. 3A) and a 57-cm capillary (Fig. 3B). Limita-

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38

ma

am Mtgrabon

7m

coo

aoa

xl00

bme (minutes)

c

I

I iso

I t4a Mgrabon

bme

I lm

I 2m

(minutes)

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 electrolyte 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 sensittvity 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 electrolyte concentration since internal heating problems increase with capillary diameter. 4. Optimize Temperature Temperature plays an important part in many separations, becauseboth solute mobility and the level of EOF are temperature-related. The majority 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 partitioning 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 wavelengths 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 shapebecomes 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.

Altria 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 seconds 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 technique, 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 secondsof 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 strategy is to minimize the sample injection time. Poor water solubility may require high levels of organic solvents. Therefore, higher sample concentrations may be appropriate. Alternatively, it may be appropriate to dissolve the sample m dilute acid or base (29) if possible. Sample stability 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

Development /Optimization

41

4 6

10 a Voltage

JI

c!ONDn-XON.9

l+!l!.L

CnpUhr&Ocm(Ld)x63Bcm~t~ x '76um (id) I 376um bd) FS Temperature= 26’C BuITer 0 03M SBS and O.OBM N&rate PA - 6 82 in 66% Waler/16% Me011 PEAK IDENTIF-ICATION l- NIACINAMIDE 2 - CYANOCOBALAhUN (812) 3 - PYRIDOXINFl HCL (BE)

26 kV

016

)(I 7s (kv)

4-NJACM 6 - TiaAMINE

01

tiCL

(B1)

16 kV

4

-A.-

1

00%

1

2

3

(4 10 kV

CI

I.

i 3

6

13

TIME

18 ( Minutes

\ 23

28

9

)

Fig. 4. Influence of voltage on resolution (From ref. Z). it is impossible to match viscosities, use of an internal standard will compensate 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 significant 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.

42

Altria

Many instruments may be operatedin constant current mode, which may be an advantageif temperaturefluctuateswithm a separationsequence.However, 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. 8. Determine

Rinse

Cycle

Selection

It is important to maintain a consistent EOF run-to-run since any variation 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 injection. 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 phosphate, it may be appropriate to rinse with 100 mk! phosphate.It is not obvious whether a rinse stepshould 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 solutions should be varied to assesstheir 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.

Development /Optimization

43

9. Optimize Injection Selection The means of sample injection should be selected and optimized for the particular application. Generally, electrokinetic sampling should only be conducted when quantifying trace levels of very mobile ions or when employing gel-filled capillaries where it is not possible to perform hydrodynamic injection. Injection times should be optimized together with sample concentration to give an acceptable peak height and shape. Generally, injection times of l-10 s may be possible. More elaborate mjection schemes employing sample stacking techniques may be employed to improve sample loading--see Chapter 16 for more details. 10. Chiral Separation Methods Development This important application areaof CE is covered in greater depth in Chapter 14.However, the following section representssomeinitial starting condition suggestionsbasedon the electrophoretic characteristics of the solute in electrolyte options l-3 (Le., low-pH FSCE, high-pH FSCE, or MECC). 10.1. LowpH FSCE This is the most commonly employed condition for chiral basic compounds. A useful starting point would be a pH 2.5 phosphate buffer containing 15 mM hydroxypropyl+cyclodextrin. The method should be developed and optimized as shown in Fig. 5. 10.2. High-pH FSCE Under high-pH conditions, the EOF sweeps along the cyclodextrin, while the anions migrate after the EOF flow. Interactions with the cyclodextrin reduce migration time. An initial electrolyte may be 15 mM borax (natural pH -9.3) containing 15 mM hydroxypropyl+cyclodextrin. Figure 6 shows a schematic for the possible optimization of a method. 10.3. MECC Combinations of SDS micellar electrolyte containing cyclodextrins have been shown to achieve chiral selectivity for both neutral and charged compounds. Optimization (Fig. 7) largely involves selection of the appropriate cyclodextrin and its concentration. 10.4. MECC for Hydrophobic Chiral Compounds For large hydrophobic compounds, it may be advisable to employ a micellar electrolyte containing bile salts, such as sodium cholate, which

44

Altria

r-l

Fig. 5. Optimization of the chiral separationof a basic compound.

Fig. 6. Optimization of the chiral separationof an acidic compound. are naturally occurring chirally selective compounds. The addition of organic solvents, such as isopropanol (IPA), can have a beneficial effect on chiral resolutions (Fig. 8).

45

Development /Optimization

Fig. 7. Optimization of the chiral MECC separation.

~~

0 ~~I~/1

0 7

&, ‘

LIzzl

~,o,~,

Fig. 8. Optimization of the chiral MECC separationfor hydrophobic compounds.

11. Method

Protocol

Figure 9 gives a detailed method protocol that, when fiAly completed, would contain all the information needed to fully document the details of a particular method.

Method no. Method purpose Method condltlons Rmse 1 Rmse 2

mm with mm with

seconds employmg

InJection parameters

Detector settmgs

. .

(equivalent to nl) nm AUFS rise time

Separate

nunutes at . ,, constant

Instrument

.

supplier

Capillary

mode model no

pm,

. cm(

. . cm todetector)

(Regenerate fresh captlkuy using 0 5M NaOH for 20 mmutes prior to use) Electrolyte composition

* .

. . .

. ... Sample composltlon

. .

. sample into

Typxal weight . Sample treatment

mgJml m .. .

.

* .

.

sonicate/filter/centfuge . .

Method development details reference

page

employmg

of

Fig. 9. Typical method protocol. 46

. ..

47

Development/Optimization References

1. McLaughlm, G. M., Nolan, J. A, Lmdahl, J. L., Morrrson, J A , and Bronze& T. J. (1992) Practical drug separations by HPCE. practical considerations. J Lzquzd Chrumatogr 15,961-102 1 2 Yeo, S K., Ong, C. P., and Li, S. F. Y (1991) Optimisation of high-performance capillary electrophoresrs of plant growth regulators usmg the overlappmg resolution mapping scheme Anal Chem. 63,2222-2225. 3. Chadwick, R. K. and Hsteh, J. C. (1991) Separation of CIS and trans double bond isomers using capillary zone electrophoresrs. Anal Chem 63,2377-2380. 4. Wemmann, W., Mater, C., Baumeister, K , Przybylski, M , Parker, C E., and Tomer K. B. (1994) Isolatton of hydrophobic hpoprotems in organic solvents by pressure assisted capdlary electrophoresis for subsequent mass spectrometrtc charactertzatton. J. Chromatogr 664,271-275 5. Sctacchitano, C. J , Mopper, B., and Specchio, J. J (1994) Identificatton and separatton of five cephalosporms by mtcellar electrokmettc capillary chromatography J Chromatogr. 657,395-399. 6. Rush, R. S., Derby, P. L., Strickland, T. W., and Rhode, F. (1993) Peptide mapping and evaluation of glycopepttde mtcroheterogenelty derived from endoprotemase digestion of erythroporetin by affimty high-performance capillary electrophorests Anal. Chem 65,1834-1842. 7. Nesi, M , Chrart, M., Carrea, G , Ottolma, G., and Rrghettt, P G (1994) Caprllary electrophorests of mcotmamtde-adenine dmucleotide and nicotmamtde-adenme dinucleottde phosphate derivatrves in coated tubular columns. J Chromatogr. 670, 215-221 8. Atamna, I. Z , Metral, C J , Muschik, G. M , and Issaq, J (1990) Factors which influence the mobility, resolutron and selectrvtty in capillary zone electrophoresls III The role of the buffer cation J Lzquzd Chromatogr 13,320 l-32 10 9. Bushey, M. M and Jorgenson, J W. (1989) Capillary electrophorests of proteins in buffers containing high concentrations of zwttteriomc salts. J Chromatogr 480, 301-3 10. 10. Altrra, K. D., Goodall D. M., and Rogan, M. M. (1994) Quantitative determinatron of drug counter-ton stolchtometry by capillary electrophoresls. Chromatographza 38,637-642. 11. Lm, Y.-M. and Sheu, S.-J. (1994) Separatron of aromatic acids by reversed electroosmottc flow capillary electrophoresis. J. Chromatogr 663,239-243. 12. Honda, S., Taga, A , Kakeht, K., Koda, S., and Okamoto, Y. (1992) Determination of cefixtme and its metabohtes by high-performance caprllary electrophoresis. J Chromatogr. 590,364-368. 13. Bullock, J. A. and Yuan, L.-C. (1991) Free solution capillary elecrophoresis of basic protems m uncoated fused-silica captllary tubing. J Micro Sep 3,241-248 14. Foly, J. P. (1990) Opttmtsatton of micellar electrokmetrc chromatography Anal Chem. 62, 1302-1308. 15. Strasters, J. K. and Khaledr, M. G. (1991) Migration behaviour of cationic solutes in micellar electrokmetic capillary chromatography. Anal Chem 63, 2502-2508.

Altria

48

16 Cole, R. 0 , Sepamak, M J , Hmze, W L., Gorse, J., and Oldlges, K. (1991) Bile salt structures III micellar electrokmetlc capillary chromatography Apphcation to hydrophobic molecule separations J Chromatogr 557, 113-123. 17 Ingvardsen, L , Mlchaelsen, S., and Sorensen, H. (1994) Analysis of individual phosphohplds by high performance capillary electrophorests J Am Od Chem Sot 71, 183-188 18 Crosby, D and El Rassl, Z (1993) Mlcellar electrokmetlc capillary chromatography wtth catlomc surfactants. J Liquid Chromatogr 16,2 16 l-2 187 19 Goebel, L. K and McNalr, H M (1991) Optlmlsatlon of resolution m mlcellar electrokinetic chromatography JHRCC 14,25 20 Terabe, S., Ishlhama, Y., Nlshl, H , Fukuyama, T , and Otsuka, K (199 1) Effect of urea addltlon m mlcellar electrokinetic chromatography J Chromatogr 545,359368 2 1 Wehr, T (1993) Recent advances in capillary electrophoresls columns LC-CG 11,

14-20. 22 HJerten, S. (1993) Electrophoreszs

14, 390

23 Smith, J. T. and Rassi, El (1993) Electrophoresrs 14,396 24 Chen, M and Cassldy, R. M (1992) Bonded-phase capdlarles and the separation of morgamc ions by capillary zone electrophoresis. J Chromatogr 602,227-234 25 Dougherty, A M , Wooley, C L , WIlllams, D L , Swaile, D F , Cole, R 0 , and Sepamak, M J. (1991) Stable phases for capillary electrophoresls J Llquld Chromatogr 14,907-912 26 Cobb, K. A , Dolmk, V , and Novotony, M (1990) Electrophoresls of protems m capillaries with hydrolytically stable surface structures Anal Chem 62, 24782483. 27 Hoffstetter-Kuhn, S , Paulus, A , Gassmann, E., and Wldmer, H M (1991) Influ-

ence of borate complexation on the electrophoretlc behavlour of carbohydrates m capillary electrophoresis. Anal Chem. 63, 1541-1547 28 Ackermans, M. T , Everaerts, F M., and Beckers, J. L (1991) Determination of some drugs by mlcellar electrokmetlc capillary chromatography The pseudoeffective moblhty as parameter for screening. J Chromatogr. 585, 123-l 3 1, 29. Altria, K. D and Chanter, Y (1993) Validation of a capillary electrophoresls method for the determination of a qumolme antlblotlc and its related impurities J Chromatogr

652,459-463

30 Coufal, P , Stulik, K , Claessens, H. A., and Cramers, C. A (1994) The magnitude and reproduclblhty of the electroosmotic flow in silica capillary tubes. JHRCC 17, 325-334.

CHAPTER5 Quantitation

Procedures

Kevin D. Altria 1. Introduction The options for conducting quantitative analysis are similar to those adopted in HPLC (I). The output format is similar to an HPLC chromatogram, i.e., a plot of UV absorbance vs time. Therefore, HPLC data handling and peak integration packages are generally applicable to CE. Main component assay may be performed using external and internal standardization or by standard addition. Impurity data can be calculated and reported as either % w/w or % area/area. Although all aspects of data handling are essentially the same as tn HPLC, it is important to normalize peak areas when calculating impurities (or enantiomers) as % area/area (2). This necessity arises smce all peaks do not pass through the detector at the same velocity. Therefore, the slower-moving peaks spend longer m the detector, giving rise to longer response times and larger peak areas.This is unlike HPLC, where all solutes are pumped through the detector at a constant flow rate. To obtain a time-independent peak area, the peak area of each peak (2,3) is divided by its corresponding migration time. The calculated areas are termed “normalized” areas. This simple manipulation is conducted by most of the commercial GE software packages on instrumentation. Calculation of results without using normalization can result in calculation of incorrect results (2). For example, a synthetic precursor to the antiulcer drug ranitidine was spiked into a solution of ranitldine at the 9.3% w/w level. The two compounds have identical UV response at the detection wavelength used for the CE separation shown in Fig. 1. From

Methods in Molecular hology, Vol 52 Cap/&y Nectrophoress Edtted by K Altna Copyright Humana Press Inc , Totowa, NJ

49

Altria

50

LlB

ClAcl AJ

40

1 l.40

Mtgratlon time (minutes)

I i6rJ

I 180

Fig. 1 Separation of a ramtldme sample spiked with a known amount of a synthetic impurity (from ref. 2)

However, the calculated % area/area indicated only 6.3% area/area impurity content. Use of normalized areas gave a result of 9.1% area/

area, confirming the splkmg level. In this example, the impurity 1sunderestimated. In the case of an impurity migrating after the main peak, it would be overestimated, 2. Main Peak Assay 2.1. External Standardization Calibration solutions of weighed amounts of the standard material are prepared and analyzed. The areas of the integrated peaks are used to calculate the response factor. Sample solutions are then analyzed, and the

recorded peak areas are multiplied by the response factor to calculate the concentration in the sample solution. Using a well-controlled validated method, it is reasonable to expect RSD values of better than l-2% for peak area (4). Typical steps are: 1. Accurately weigh and transfer a portion of the reference material and samplesto appropriatevolumetric flasks

Quantitation

Procedures

51

2. Dilute to volume with the dissolving solvent. 3 Shake or somcate as required to ensure dissolution of the maternal. 4. Falter or centrifuge the sample tf requu-ed, and transfer the sample solution to autosampler vials 5. Place the vials contammg the electrolyte, rinse solution(s), calrbrations, blank, and samples mto the approprtate positions on the autosampler. 6 Perform a test injection of a test mixture to confirm the system 1soperating correctly, 7. Analyze the samples under the conditions specified m the method. A typical sequence will contain analysis of a solution of the dissolving solvent, and a number of injections of the calibration solutions and sample solutions. An appropriate sequence for four samples may be: 1. 2 3. 4. 5 6 7. 8. 9. 10. Il.

Blank. Calibration Calibration Sample 1 Sample 2 Calibration Calibration Sample 3. Sample 4. Calibratton Calibratron

1. 2. 1. 2 1. 2.

Each solution should be injected in duplicate resulting in 22 injections for this example. The integrated peak areas for the calibrations are then used to calculate the response as given below: Response factor = [weight calibration (mg) x % purity / (1) peak area x dilution volume (mL) x 1001 The response factors for all the calibration solutions are calculated and averaged to give a Mean Response Factor (MRF). The concentration of analyte in the sample, if supplied as a solution, is calculated by: mg/mL = peak area x MRF

(2)

If a solid sample was sampled, then the % w/w purity is calculated as: % w/w = [MRF x peak area x dilution volume (mL) / wt (mg)] (3) An example of the use of an internal standard is shown in Fig. 2 (5) m the determination of the drug sumatriptan in injection solutions. The use of

Altria

52

3.00

Retention

5.00

7.00

8.00

time h mhutee

Fig. 2. Separation of sumatriptan employmg an internal standard (from ref. 5). an internal standard allowed an average RSD value of < 1.O%for response

factors to be obtained. 2.2. Internal

Standardization

Steps l-7 (see Section 1.1.) for external standardization are followed, replacing the dissolving solvent by a solution of an appropriate internal standard. The concentration of the internal standard in the final sample solution should approximately match that of the sample. Calculations are modified to incorporate the internal standard. Peak area ratio (PAR) = (peak area sample / peak area mtemal standard)

(4)

Response factor = [wt (mg) x % purity / PAR x dilution volume (mL) x 1001

(5)

Solution concentration = PAR x MRF % w/w = [PAR x MRF x dilution volume (mL) / wt (mg)] 2.3. Standard Addition

(6)

This calibration procedure involves addition of known amounts of standard to the sample solutions (6). Thesespiked samplesare then analyzed and

Quantitation

53

Procedures

the peak areas are plotted against spiking level. The sample content is then calculated from the intercept and slope of the line. The sample spiking can be performed manually or can be preprogrammed into the separation method. 2.4. Manual

Standard

Addition

This procedure (6) is of particular benefit when dealing with samples of varying viscosity, such as biofluids. Since injection volumes are related to sample viscosity, it is essential to match closely the viscosity of the samples and calibration solutions. 1. Takealiquotsof the samplesolutions,typically four, into appropriate vessels. 2. Prepare four calibratron solutions over the required calibration range. 3. Add a consistent volume of each of the standard solutions to the sample solution (for example, add 1.O mL of standard solution to 10.0 mL of sample ahquot). 4. Analyze, at least in duplicate, each of the spiked samples and an unspiked sample. 5. Plot the peak areas agamst spiking level (typlcally recorded m mg/mL or mg addition). 6. Using the slope and intercept values, calculate the sample concentration. A typical calculation is shown below where spiking level IS recorded as mg: mg/mL = [intercept x sample volume (mL) / slope] (7) This approach is demonstrated in Fig. 3 (6), where standard addition is

used to determine trace levels of fluoride. 2.5. Automated

Standard

Addition

In this approach (7), two injection steps are incorporated into the separation method as shown below: Rinse 1: Xmin Rinse 2: Y min Set detector: desired 1 InjectIon 1: x s from standard Injection 2: x s from sample

Separate:requiredmin at selectedvoltage To cover a calibration range, sampling times should be varied appropriately. For example, the time for injection 2 could be set at 5 s, and sampling times for injection 1 set at 0, 1,3, and 5 s. This approach is of use when quantifying a component in a complex mixture, as component identification is simultaneously confirmed. Fig. 4A shows separation of

Altria

54 1600. 1400

-

1200

-

lcaO800

-

600400

-

200

0,. 0.0

, 0 2

I‘

0.4

) 0.6

.

I 0.8

'

I 1.0

-

J 1.2

Fluoride Concentration Ftg. 3. Standard addition graph for fluoride deterrnmatton (from ref. 6). a 5-s injection of a solution of a pharmaceutical. Fig. 4B shows a 5-s injection of a solution of a pharmaceutical followed immediately by a 5-s mjection of a solution of a known impurity. It can be clearly seen which peak in Ftg. 4A is attributable to the specified impurity.

3. Impurity

Contents

Impurity levels may be quoted as % w/w (8) or % area/area (9,10) depending on the application. The impurity level may be calculated as % w/w if an appropriate standard of the impurity is available (8). More commonly, impurity results are calculated as % total area, since standards for all impurities may not be available.

3.1. Percent

(wlw) Impurity

Determination

Procedure

1 Prepare impurity caltbratton soluttons of approprtate concentratton to match likely levels present in samples. 2. Prepare sample soluttons rn the same dissolving solvent as the standards. 3. Analyze both samples and standards in an injection sequence. 4. Calculate impurity levels using response factors from calculatton soluttons. RF = [wt calibration (mg) x % purity / dilutton volume (mL) x 1001 (8)

Quantitation

3 c i5 m 88

Procedures

55

B.OO-

B.OoII 7.00-

pp

6 lO.OO-

B.OD-

I

8.at3-

6.0

Ill

II

1 8.00

7.00 Migrallon

time

8.00

(mmutes)

Fig. 4. Separation of salbutamol tmpurities (from ref. 7). (A) 5-s injection of salbutamol solution; (B) 5-s injection of salbutamol solution + 5-s injection of salbutamol tmpurlty solution.

Altria % w/w = [MRF x peak area x dllutlon volume x 100 / wt sample (mg)] (9)

This approach has been employed for the determination of the dimeric impurities of salbutamol(8). Fig. 4A shows the separation. The calculated results in % w/w were then directly compared to HPLC and TLC results. 3.2. Percent

Area/Area

Impurity

Procedure

This procedure 1smost commonly employed for ease of operation and simplicity (9). In addition, isolated standards of all impurities may not be available. This procedure infers that all impurities have similar response factors at the detection wavelength to the main component. If the impurity response factors differ significantly, appropriate adjustment of data prior to reporting is necessary 1. Analyze sample solutions and a blank at least m duphcate. 2. Integrate all sample related peaks, ignoring all peaks attributed to the blank. 3. Normalize each peak area to Its correspondmg mlgratlon time: Normalized peak area = (peak area / migration time) (10) 4. Add all the normalized peak areas to give a total normalized area for the electropherogram. 5. Calculate specific impurity levels as: % area = (normalized peak area x 100 / total normahzed areas) (11) 6. If the identity of a peak 1sunknown, it 1soften useful to calculate the relative migration time (RMT) as an identifier. RMT 1scalculated by* RMT = (migration time Impurity / migration time of main component) (12)

Impurity profiles for the duplicate injections of each sample solution should be compared. If the profiles do not differ significantly, impurity results for either injection or an average can be quoted. Typical reporting format may include: No. of lmpurltles Total % area lmpurltles Greatest impurity (% area) Identity of greatest impurity Second greatest impurity (% area)

Identity of secondgreatestimpurity Figure 5 shows the separation of related impurities in salicylamide by MECC (9), which were calculated as % area/areawith a detection limit of 0.999 were obtained. The limit of detection was 5 pg/mL. A range of 10 chlorophenoxy acids were baseline-separated employmg MECC conditions within a 9-min analysis time (66). Injection repeatability was 1% for migration times and 2% for peak areas. The separation of eight sulfonylurea herbicides has been achieved (64) using an ammonium acetate:acetonitrile 75:25 electrolyte adjusted to pH 5.0 with acetic acid. The baseline-resolved components were detected by both UV and a mass spectrometer (MS) (64). An MS-MS arrangement was also employed to obtain spectral data for the peaks, which were matched against library spectra of authentic samples. Levels of sulfonylureas were spiked into soil extract samples and were determined by the method. To date, only one report has focused on the application of CE to industrial samples (63). In this paper, the use of CE was shown to monitor impurity levels in phenoxyacid herbicides. A range of herbicides and

Additional

Application

Areas

325

related impurities were separatedusing a lithium acetate buffer at pH 4.8 with detection at 200 nm. Various cyclodextrins were added to appropriately modify selectivity. Figure 9 shows separation of a test mixture (Fig. 9A) and a production batch (Fig. 9B) employing dimethyl-P-CD as electrolyte modifier. Certain impurities are chirally resolved under these conditions, Detection levels of CO.1% were possible for the impurities. Good precision of peak area (I. 1-l .4% RSD) and migration time (0.3-0.5% RSD) was achieved with linearities of CO.9998. Chiral analysis of herbicides produced as single enantiomers was performed. The CE method gave good agreement with results generated by HPLC test methods. The aqueous stability of metsulfuron has been monitored by CE (67). Three major degradation products were produced following storage for 4 d. Peak identification was confirmed by GC-MS. Another important analytical application in agrochemical analysis is the determination of pesticide residues. Levels of the sulfonylureas, chlorsulfuron, and metsulfuron have been determined in tap water at subppb levels (68). Samples were prepared following solid-phase extraction and analyzed using a pH 9.0 borate buffer with detection at 214 nm. Recoveries of over 90% were demonstrated. In a similar paper, four herbicides were determined by MECC in tap and drinking water (67). Sample preparation involved a lOOO-fold sample enrichment followmg solid-phase extraction (67). Good (80-95%) recoveries were shown in the range l-5 p.g/L range. Levels of paraquat and diquat were determined in crop water by both HPLC and CE (69). Similar detection limits were obtained for the two techniques. 4. Carbohydrates Carbohydrate analysis representsa particular challenge to CE in that the principal analytes possess no readily ionizable groups or chromophores. Three approaches have been suggested to overcome those difficulties: 1. Indirect detectionat htgh pH; 2. Borate complexation with detectionat 195nm; and more commonly 3. Separationof chargedcarbohydratederivatives. Operation at pH values of approx 12 ensures the ionization of the weakly acidic hydroxyl groups on underivatized saccharides (70). Sorbic acid was added at 6 mM to provide the background UV signal for indirect detection.

124

0

120

0

A

0

F CPA

t 116

0 b

I -KPP

OP

A

HO

CP

Ir

109

5

’

B

Pi

108 0 E 106

5

t t

Pechn.

WCPP

H

HO

i-ktCPP YCPA

A 100

5 --Iryw*h*iJ

99OL

' 2 0

3

"

' 3.0

5

I

' * 4 0

e

I

" 5.0

I 6.0

I Tim0

I

I

I, 1.0

I

,I,,,,,,,,, 8.0

9.0

10.0

11.0

12

0

Iminutes)

Fig. 9. Separation of a test mixture: (A) a production batch (B) and employmg dlmethyl-@-CD as modifier Reprinted with permission from ref. 63. Separation condltlons: 20 g/L mM dimethyl+-cyclodextrin m 30 mM hthium acetate (PH 4.Q detection: 200 nm.

326

Altria

Many carbohydrates can complex m solution with borate ions, which permit their separation as anions, The complexation 1sfavored at higher temperature and higher borate concentrations (72). Underivatized carbohydrates were resolved using 60 mM borate at 60°C. A number of derivatizing agents have been suggested, and the selection should be made on the functionality present on the carbohydrate of interest. For example, CBQCA is a useful choice for carbohydrates containing amino groups (72). Perhaps the most appropriate of all the available derivatives is 8-amino-napthalene- 1,3,6-trisulfonic acid (ANTS) (73). ANTS derivatives can be prepared relatively simply and separated over a wide pH range, since the ANTS molecule possessesthree acidic groups with varyingp&s. The presence of the napthalene group ensures good UV and fluorescence sensitivity. Aminobenzoate derivatives are a useful alternative (74), particularly for ketoses. Separation of aminobenzoate derivatives (Fig. 10) has been performed at pH 10.0 with electrolytes containing high levels of borate (74). 5. Vitamin Analysis This area has yet to receive extensive development, although the applicability of CE to the analysis of vitamins has been clearly demonstrated (7.5-80). This is somewhat surprrsmg given the extensive development and application of CE to analysis of pharmaceuticals. The range of vitamins can be broadly subdivided into water- or oilsoluble. It is possible to perform separation of water-soluble vitamins using simple free solution CE, whereas use of MECC is mandatory for the analysis of oil-soluble vitamins. The most detailed study on vitamin analysis used both FSCE and MECC to analyze a range of vitamins m commercial vitamin preparations (7.5). Table 6 shows the FSCE and MECC results compared to results generated by the USP HPLC method. Acceptable precision data of 122 0 f 2.2 Soft capsule Bl (10 mg) PP (30 mg) 111 3 + 1.8 112 I +3 1 B2 (7 mg) 1086k 1.8 B6 (5 mg> Repnnted wtth pernnsslon from ref 75 n/a = not analyzed Tablet

MECC

HPLC

1236_+26 108.0 X!I1 2 994+2 1 113 7* 17 112.4 3~ 1 3 1094+09 119.3 + 2.9 106 2-1: 3.1 126.6 21 1 7 1086k 17 1149k 16 108 2 Z!I 1.6

1238+36 108752 1 1039+07 1124+3 2 111.6+ 16 1115+34 117.2 + 2 2 1132+39 n/a n/a n/a n/a

solid-phase extraction (84). However, the rugged nature of the CE capillary format can also allow direct mjection of biofluids, such as serum (90) and urine (86), with obvious savings in analysis time and cost of consumables. The majority of direct injection analyses are performed by MECC, since the SDS micelles strongly interact with the sample proteins causing the proteins to be eluted later, and the small solutes of interest are then visible free from protein interfaces. Figure 11 shows that antiepileptic drugs can be directly monitored in patient serum as the proteins migrate after the peaks of interest (90). The performance of CE methods in clinical assays has been assessed by many workers and general comments indicate that CE is not as sensitive as HPLC, but has benefits in terms of simplicity and possible sample pretreatment reductions. Validation parameters, such as linearity, recoveries, and precision, show acceptable performance (81,83,86). Crossvalidation of CE results with other techniques, such as HPLC (86) and immunoassays (90,91), show that CE is capable of generating accurate results. Table 7 compares levels of creatinine and uric acid as determined by both an enzymatic method and by MECC (92). Despite the interest and research focus in this area, increased routine application may require

Additional

Application

Areas

331 Proteins

Phenobarbital 1,. 5

.,

10

15

.

2( 1

Fig. 11. Separation of antieplleptic drugs by direct qectlon of patient serum. ReprInted with permission from ref. 90. Separation conditions: 75 n-&I SDS, 6 mA4 borax, 10 mA4 phosphate, 220 nm, 35°C.

Comparison

Table 7 of Levels as Determmed by MECC and Enzymatic Methods Creatmine, yglmL

Plasmasample I 2 3 4 5 6 7 8

Uric acrd, pg/mL

Enzymatic

MECC

5 7 4 6 7 1 8 4

5 7 4 5 7 N/R 8 4

Enzymatic 28 50 38 27 66 12 56 60

MECC 32 51 N/R 32 66 11 58 60

Reprinted with permIssIon from ref 92 N/R = no result obtamed

further advances in sensitivity by instrument improvements sample introduction procedures.

or by better

7. Alternative Detection Systems (Including CE-MS) Currently, all commercial CE systems incorporate a UV-absorbance detector. Some systems are modular, or partially modular, and alternative detection systems can be employed. The principal detection alternatives to UV-absorbance detectlon that are commercially available are fluorescence and mass spectrometry. Other less-developed detector

Altria

332

options include electrochemical (93,94) and conductivity detection (9.5). An increasing number of commercial instruments are also available with UV-absorbance photo-diode array facilities. Diode array detectors have been shown to be of use in peak identification and peak purity assessments (96,9 7). 7.1. CE-Mass

Spectrometry

There has been a great deal of activity in the area of interfacing CE to mass spectrometers, and the advances to date have recently been reviewed (98). A number of interfaces have been successfully demonstrated, include continuous-flow fast atom bombardment (99), electrospray (100, 10 I), and ionspray (102). Generally, reports have indicated that CE-MS is less sensitive than UV-absorbance detection, which may have limited a more substantial exploitation of this detection mode. However, the significant potential sensitivity advances offered by ion-trap MS (103) or the use of on-line stacking in CE (104) may lead to an alleviation of this issue. 8. Amino Acid Analysis Much of the early development work in CE was performed using derivatized amino acids as test solutes. A range of derivatization agents are avaliable for this purpose (Table 8). The vast majority of separations have been achieved employing SDS-based MECC conditions with the addition of organic solvent (105). Figure 12 shows the highly efficient resolution of 23 dansylated amino acids using 102 mMSDS, pH 9.2, electrolyte and a temperature of 10°C (105). Underivatized amino acids have not been widely analyzed, since they generally possess very limited chromophores. However, native amino acids have been separated and monitored by indirect detection, employlng 1 mM sodium salicylate at pH 9.7 (11.5). 9. Particulates,

Bacteria, and Dyes 9.1. Analysis of Particulates CE has been successfully applied to several separations of large polymeric species, such as polysterene latex particles (I I6) and Jeffamine polymers having molecular weights up to 2100 (II 7). Separations are performed in free solution with resolutions owing to size differences. Silica gel sols, which are used in the preparation of HPLC packing mate-

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333

Table 8 Derwatlzatlon Reagents Employed m Amino Acid Analysis Derwatwe

Reference no

PTH FITC CBQCA TBQCA Dns OPA NDA

106 107,108 109,rro Ill 112,105 113 113,114

PTH (phenythlohydantom), FITC (fluorescem lsothlocyanate), CBQCA (3-[4-carboxybenzoyll-2-qumolmecarboxaldehyde), Dns (Dansyl), OPA (o-phthaladehyde), TCQCA (3-[4-tetrazolebenzoyll-2-qumolmecarboxaldehyde), NDA (napthalene dlaldehyde)

0

10

12

14

16

7.0

22

mln

Fig. 12. Resolution of 23 dansylated amino acids usmg MECC at 10°C Reprmted with permlssion from ref. 105. Separation conditions: 20 mA4borax, 100 mMSDS, 10°C, 214 run. rial, have also been characterized by CE (118) using pH 9.0 buffers and detection at 190 nm. Silica sol colloids up to 500 nm diameter were separated (118).

of Bacteria by CE Mixtures of various viable bacteria, such as Enterococcus, Streptococcus, and Staphylococcus strains, were resolved by CE (129) using Tris/Borate/EDTA buffer. The pH of this buffer is high, and the bacteria 9.2. Separation

and Isolation

were resolved as anions. UV detection at 190 or 200 nm, together with

use of a loo-pm capillary, allowed sufficient sensitivity. Preparative CE was performed to collect specific bacteria from mixtures. Positive iden-

Al tria

334

tification of collected fractions was achieved by several techniques, including metabolic fermentation. Purities of recovered fractions were >99%. The authors concluded that CE could afford the microbiologist a new tool for studying the composition and distribution of microorganisms in mixed populations. It 1s noted that these separations were conducted on homemade equipment and that sophisticated commercial equipment may offer significant advantages in terms of improved performance and sensitivity.

9.3. Determination

of Dyes by CE

Currently HPLC IS predominantly employed in the separation and determination of levels of cationic, anionic, and neutral dyes, and dye intermediates. CE has been shown to be of use in this area (120,121). Many dyes have two or three membered ring structures and are water soluble, making them very suitable for analysis by CE.

Notes Added

in Proof

1. Analysis of small ions by capillary electrophoresls: An optrmized separatlon has been reported (122) that allows simultaneous determmatlon of ammonium, alkali, alkalme-earth, and various transition metals using an electrolyte containing lmidazole, crown ether, methanol, and HIBA Low ppb detection levels were possible with electrokinetic mJectlon. Laserinduced indirect fluorlmetrlc detection of cations has been reported (123); the electrolyte employed contained fluorescem sodium and EDTA, and low-mid ppb levels of metal ions could be detected using pressure mJectlon. Recent advances in the determination of anions has centered on the optlmlzation of electrolyte systems, For example, the use of p-aminobenzoate as an electrolyte additive has been shown (124) to be useful for the detection of orgamc acids. 2,6-napthalene dicarboxylic acid has been employed as a UV absorber and has been shown (125) to be a considerable improvement over pthalate. Migration time drifts can occur using the standard electrolyte containing chromate because of electrolyte depletion. This problem can be overcome (126) by the addition of 1 r&k! 5,Sdiethylbarblturate to the electrolyte. 2. Experimental design: Multivariate regression analysis has been employed to study the effect that various ratios of EDTA and borate concentrations have on the migration time of metal complexes (127). A central composite design has been utilized for optimization of electrolyte composition (122). A full factorial design was then used to measure the mam effects of several parameters on the EOF velocity.

Additional

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Carbohydrate analysis: A recent survey (128) has been published concernmg the application of CE to carbohydrate analysis. It comprehensively reviews the derivative types available and a range of appltcations. Another recent report (129) dlscussed the use of tags, such as ANTS Underlvatized carbohydrates have been separated using NaOH electrolytes with electrochemical detection (130). Indirect UV detection using a pH 12.3 electrolyte containing sorbic acid has been used (131) to assay the carbohydrate content m fruit Juices and good agreement with HPLC data was obtained 4. Vitamin analysis: Surpnsmgly, there contmues to be relatively few reports of analysis of vitamins by CE. One notable exception is the work concernmg the determmatlon of vitamin A in dried blood spots. Laser-based fluorescence measurements allowed (232) a detection limit of 3 pg/L to be obtained for retinol. The analysis could be conducted from one or two drops of blood. 5. Biomedical apphcations: The number of biomedical apphcatlons of CE contmues to expand rapidly. Some particular examples are discussed covering both applications and methodology approaches. Theophylline and metabolites have been determined in urine (133) using solld-phase extraction pretreatment. A variety of ephedrine alkaloids were determined m urine with direct sample injection (234), and levels of free and total 7-hydroxy-coumarin were determined (23.5) in both urine and serum samples. The use of SDS solution as a rinse solution between analyses of blosamples has been shown to be more effective (136) than conventional rinsing regimens. The different approaches to quantifying drug m human serum followmg direct sample Injection have been compared (23 7) 6. CE-MS: The apphcatron of CE-MS combinations as separation-detectlon systems continues to grow. For example, peptides (138) and DNA fragments (139) have been detected by MS followmg their separation by CE. 3.

References 1. Beck,W. andEngelhardt,H. (1992) Capillary electrophoreslsof orgamcand morganlc cationswith mdlrect UV detection.Chromatographza 33,3 13-3 16. 2. Chen, M. andCassldy,R M. (1993) Separationof metal ions by capillary electrophoresis.J Chromatogr 640,425-43 1 3. Weston,A., Brown, P. R., Heckenberg,A , Jandlk, P., and Jones,W. R. (1992) Effect of electrolyte composltionon the separationof morgamc metal catlonsby capillary ion electrophoresls.J Chromatogr 602, 249-256. 4. Shi,Y. andFritz, J. S.(1993) Separationof metal ionsby capillary electrophoresls with a complexlng electrolyte.J Chromatogr 640,473-479. 5. Weston,A., Brown, P. R., Jandlk, P , Jones,W R., andHeckenberg,A. L (1992) Factors affecting the separationof inorganic metal cations by capillary electrophoresis.J. Chromatogr 593,289-295.

Altria 6 Jackson, P E and Haddad, P (1993) Capillary electrophoresis of inorganic ions and low-molecular-mass iomc solutes TRAC 12,231-238. 7 Quang, C and Khaledi, M G (1994) Prediction and optimisatton of the separation of metal cations by capillary electrophoresis with indirect UV detection J Chromatogr 659,459-466 8 Shi, Y. and Fritz, J S (1994) New electrolyte systems for the determmation of metal cations by capillary zone electrophorests J Chromatogr 671,42%-435 9 Backmann, K , Boden, J., and Haumann, I. (1992) Indirect fluorimetric detection of alkah and alkaline earth metal ions m capillary zone electrophoresis with cermm (III) as carrier electrolyte. J Chromatogr 626,259-265 10. Altrta, K D., Goodall, D M , and Rogan, M M (1994) Quantitative determmatton of drug counter-ton stoichiometry by capillary electrophoresis Chromatographla 38,637-642 11 Swartz, M E. (1993) Capillary electrophoretic determmation of morgamc tons in prenatal vitamin formulation J. Chromatogr 640,44 l-444 12 Koberda, M., Konkowskt, M , Youngberg, P., Jones, W. R , and Weston, A (1992) Capillary electrophoretic determination of alkali and alkaline-earth cations in various multiple electrolyte solutions for parenteral use J Chromatogr 602,235-240 13. Morawski, J , Alden, P , and Sims, A. (1993) Analysis of cationic nutrients from foods by ion chromatography J Chromatogr. 640,359-364 14 KaJiwara, H , Sato, A , and Kaneko, S. (1993) Analysis of calcmm and magnesium ions in wheat flour by capillary zone electrophoresis BIOSCL Blotech, Bzochem 57, lOlO,lOll. 15 Motomizu, S , Oshima, M., Matsuda, S -Y., Obata, Y., and Tanaka, H (1992) Separation and determmatton of alkaline-earth metal ions as UV absorbing chelates with EDTA by capillary electrophoresis. Determmation of calcium and magnesium m water and serum samples. Anal Scz 8,619-624 16 Buckberger, W , Semenova, 0 P., and Timerbaev, A R. (1993) Metal ton captllary zone electrophoresis with direct UV detection. separation of metal cyanide complexes JHRCC 16, 153-156. 17 Aguilar, M., Farran, A , and Martinez, M. (1993) Determmatton of gold (1) and silver (1) cyanides m ores by capillary zone electrophoresis. J Chromatogr 635, 127-13 1 18 Swaile, D. F and Sepamak, M J (199 1) Determmatton of metal ions by capillary zone electrophoresis with on-column chelation usmg 8-hydroxyquinoline-%sulfomc acid. Anal Chem 63, 179-184 19. Timerbaev, A. R , Buchberger, W , Semenova, 0 P., and Bonn, G K (1993) Metal ion capillary zone electrophorests with direct UV detection determination of transition metals using a 8-hydroxyqumoline-5-sulphomc acid chelating system. J, Chromatogr 630,379-389 20 Pretswell, E. L , Morrisson, A R., and Park, J S. (1993) Compartson of capillary zone electrophoresis with standard gravimetric analysis and ion chromatography for the determination of inorganic amons in detergent matrtces. Analyst 118,1265--1267. 21 Harrold, M. P., Wojtusik, M. J , Riviello, J , and Henson, P. (1993) Parameters mfluencmg separation and detection of anions by captllary electrophorests, J Chromatogr 640,463-47 1

Additional

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22. Kaneta, T., Tanaka, S., Taga, M , and Yoshrda, H (1992) Migration behaviour of inorganic amens m micellar electrokmetrc capillary chromatography using a catiomc surfactant. Anal Chem 64,798-801 23. Jones, W. R. (1993) Method development approaches for capillary ion analysts. J. Chromatogr. 640,387-395. 24. Kelly, L. and Nelson, R. J. (1993) Capillary electrophoresrs of organic acids and anions J. Liquid Chromatogr 16,2 103-2 112 25. Buchberger, W. and Haddad, P. R (1992) Effects of carrier electrolyte composrtion on selectrvrty m capillary zone electrophoresrs of low-molecular-mass anions. J Chromatogr 608,.59-64. 26. Ackermans, M. T., Ackermans-Loonen, J C. J M., and Beckers, J L (1992) Determmatton of propronate in bread using capillary zone electrophorests J Chromatogr 627,273-279 27. Jackson, P. E. and Haddad, P. (1993) Capillary electrophoresrs of inorganic ions and low-molecular-mass tonic solutes. TR4C 12,231-238. 28. Tinfdall, G. W., Wilder, D. R., and Perry, R. L. (1993) Optrmtsmg dynamic range for the analysts of small ions by capillary zone electrophoresrs. J Chromatogr 641, 163-167. 29. Ryder, S. (1992) Determination of sodmm vinyl sulphonate m water-soluble polymers using capillary zone electrophoresis J Chromatogr. 605, 143-147. 30 Wildman, B. J., Jackson, P E., Jones, W. R., and Alden, P G. (1991) Analysis of anion constituents of urine by inorganic caprllary eiectrophoresrs. J. Chromatogr. 546,459-466 3 1. Romano, J. P and Krol, J (1993) Capillary ion electrophoresrs, an envuonmental

method for the determmatron of amons in water J. Chromatogr 640,403-4 12. 32. Jackson, P. E and Haddad, P. R. (1993) Optrmrsation of inJection technique m capillary electrophoresrs for the determmatron of trace levels of anions in envrromental samples. J Chromatogr. 640,481-487. 33 Jandrk, P and Jones, W. R (1991) Optimisatron of detection sensitivity m the capillary electrophoresis of inorganic anions. J. Chromatogr 546,43 l-443 34. Salomon, D R. and Romano, J. (1992) Applications of capillary ion electrophoresis in the pulp and paper industry. J Chromatogr. 602,2 19-225. 35 Hargadon, K. A. and McCord, B. R. (1992) Explosive residue analysis by caprllary electrophoresis and ion chromatography J. Chromatogr. 602,241-247 36. Chadwick, R. C and Hsreh, J C. (1991) Separation of cis and trans double bond isomers using caprllary zone electrophoresis Anal Chem 63,2377-2380 37. Ng, C. L., Lee, H. K., and LI, S F Y. (1992) Analysis of food additives by ronpairing electrokmetic chromatography. J. Chrom Sci 30, 167-l 70. 38. Vindevogel, J. and Sandra, P. (1991) Resolutron optrmisatron in mrcellar electrokinetic chromatography: use of Plackett-Burman statistical design for the analysis of testosterone esters. Anal Chem. 63, 1530-l 536. 39. Rogan, M. M., Altna, K D., and Goodall, D. M. (1994) Plackett-Burman experimental design m chiral capillary electrophoreus. Chromatographia 38,723-729. 40. Ng, C. L., Lee, H. K., and Li, S F Y. (1993) Systematic optimisatron of capillary electrophoresis of sulphonamides. J. Chromatogr 598, 133-I 38.

Altria 4 1 Ng, C. L , Ong, C P , Lee, H K , and LI, S F Y (1992) Systematic opttmrsatron of mlcellar electrokmetrc chromatographrc separation of flavanords. Chromatographla 34, 166-172 42. Ng, C L., Toh, Y. L , Lr, S. F Y., and Lee, H. K (1993) Captllary electrophoresrs of btologtcally important compounds. opttmlsatron of separation condmons by the overlapping resolution mapping scheme J Lrqurd Chromatogr 16, 36533666. 43 Yeo, S. K , Ong, C. P., and Lr, S. F Y. (1991) Optrmrsatron of high-performance capillary electrophoresrs of plant growth regulators using the overlapping resolution mapping scheme Anal Chem 63,2222-2225 44 Altrta, K D. and Filbey, S D. (1994) The applrcatron of experimental design to the robustness testing of a method for the determmatton of drug related tmpurmes by capillary electrophoresrs Chromatographla 39,306-3 10. 45. Frlbey, S. D and Altria, K D. (1994) Robustness testing of a capillary electrophorests method for the determmatron of potassium content in the potassium salt of an acidic drug. J Capdlary Electrophoresls 1, 190-195. 46 Castagnola, M., Rossettr, D. V , Casstano, L., Rabmo, R., Nocca, G , and Gtardma, B (1993) Opttmrsatton of phenylhydantomammo acid separation by micellar electrokmettc capillary chromatography. J Chromatogr 638, 327-334 47 Vanbel, P F , Gilhard, J A., and Tilqum, B. (1993) Chemometric opttmrsatton m drug analysis by HPLC: a crmcal evaluation of the quality criteria used m the analysis of drug purity. Chromatogruphm 36, 120-l 24 48 Andersson, A M , Karlsson, A , Josefson, M., and Gottfries, J. (1994) Evaluatton of mobile phase additives m LC-systems using chemometrrcs. Chromatographla 38,715-722. 49 Rrghezza, M. and Chretren, J R. (1993) Factor analysis of experimental design m chromatography. Chromatographza 38, 125-129. 50 Mullholland, M. and Waterhouse, J. (1988) Investrgatron of the limttatrons of saturated fractional factorial experimental desrgns, with confounding effects for an HPLC ruggedness test Chromatogruphza 25, 769-774. 5 1 Mullholland, M (1988) Ruggedness testing in analytical chemistry TRAC 7, 383-389 52 Berrrdge, J. C. (1989) Chemometrrcs and method development in hrgh-performance hqurd chromatography. Part 2 sequential experimental designs Chemometrlcs Intell Lab Syst 5, 195-207 53. Lmdberg, W and Johannson, K. (1981) Apphcatron of stattstrcal optimrsatron methods to the separation of morphine, codeme, noscapine and papaverme m reversed-phase ion-pair chromatography. J Chromatogr. 211,20 l-2 12 54. Ahmad, S. U , Lane-Cam, C. A , and Bolton, S M. (1990) Factorial design in the study of the effects of selected liquid chromatographic conditions on resolution and capacity factors J Liquid Chromatogr 13,525 55 Plackett, R C. and Burman, J. P (1946) The desrgn of optimum multtfactorral experiments. Blometrrca 23,305-325. 56. Demmg, S. L. and Morgan, S. L (1983) Teaching the fundamentals of expenmental design. Anal Chum Acta. 150, 183-198.

Additional

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57 Box, G. E P., and Hunter, J S. (1978) m Statlsttcsfor Experiments, An Introduction to design, Data Analysis and Model Bulldmg, Wiley, New York, pp 291453. 58. Tucker, R. P., Fell, A. F., Berndge, J C., and Coleman, M W (1992) Computeraided models for optimisation of eluent parameters m chnal hquid chromatography Chtrality 4,3 16-322 59 Yao, Y C., Lee, H. K , and LI, S F Y. (1993) Opttmrsatron of mobile phase composition for HPLC separations of mtroaromattcs using overlappmg resolutron mapping. J Llq Chromatogr 16,2223-2225 60. Glajch, J. L , Kirkland, J S , Squire, K M., and Mmor, J M (1980) Optimization of solvent strength and selectivity for reversed-phase hqurd chromatography using an inter-active mixture-design statrstrcal technique J Chromatogr 199, 57-79 61 Mullholland, M and Waterhouse, J (1987) Development and evaluation of an automated procedure for the ruggedness testing of chromatographtc conditions m high-performance hqurd chromatography J Chromatogr 395,539-55 1 62. Thomas, B R. and Ghodbane, S. (1993) Evaluation of a mixed micellar electrokinetic capillary electrophoresis method for validated pharmaceutical quahty control J. Liquid Chromatogr 16, 1983-2006. 63 Nielen, M W. F (1993) (Enantto-) separation of phenoxy acid herbicides usmg capillary zone electrophoresis J Chromatogr 637, 8 l-90 64 Garcia, F. and Hemon, J (1992) Fast capillary electrophoresis-ion spray mass spectrometric determinatton of sulfonylureas. J Chromatogr 606, 237-247 65 Pianettr, G. A , Tavema, M., Baillet, A , Mahuzler, G., and Baylocq-Ferrrer, D (1993) Determmatton of alkylphosphomc acids by capillary zone electrophoresrs using indirect UV detection J Chromatogr 630,37 l-377 66 Wu, Q., Claessens, H. A , and Cramers, C. A (1992) The separation of herbicides by micellar electrokmetic capillary chromatography Chromatographta 34,25-30. 67. Dinelh, G., Bonetti, A , Catizone, P., and Gallettt, G C. (1994) Separation and detection of herbicides m water by mrcellar electrokmetrc capillary chromatography J Chromatogr 656,275-280 68. Dmelli, G , Vicar-i, A , and Catrzone, P (1993) Use of captllary electrophoresrs for detection of metsulfitron and chlorsulfuron m tap water. J. Agrlc Food Chem 41,742-746

69 Camerro, M C., Puignou, L , Galceran, M. T. (1994) Comparison of capillary electrophoresis and reversed phase ion-pair high-performance hqurd chromatography for the determination of paraquat, diquat and drfenzoquat. J. Chromatogr 669,2 I7-224 70. Vorndran, A. E , Oefner, P. J , Scherz, H , and Bonn, G. K (1992) Indirect UV detection of carbohydrates m capillary zone electrophorests. Chromatographta 33, 163-l 68. 71. Hoffstetter-Kuhn, S , Paulus, A , Gassman, E., and Wrdmer, H. M. (1992) Influence of borate complexation on the electrophoretrc behaviour of carbohydrates m capillary electrophoresis. Anal Chem 63, 154 l-l 547 72. Lm, J., Shirota, O., and Novotny, M. (1991) Capillary electrophoresis of amino sugars with laser-induced fluorescence detection. Anal Chem. 63,4 13-4 17.

340

Altria

73 Chtesa, C and Horvath, C S. (1993) Captllary zonal electrophoresis of maltoohgosacchartdes dertvattsed wtth 8-ammonapthalene-1,3,6,-trtsulphonic acid. J Chromatogr

645,337-352

74. Grill, E , Huber, C , Oefner, P , Vordran, A., and Bonn, G. (1993) Captllary zone electrophorests of p-aminobenzotc acid derivatives of aldoses, ketoses and uranic acids Electrophoresrs 14, 1004-1010 75 Boonkerd, S , Detaevernter, M. R , and Michotts, Y (1994) Use of capillary electrophoresis for the determmation of vttamms of the B group m pharmaceuttcal preparations. J Chromatogr 670,209-2 14 76 Ong, C P , Ng, C. L , Lee, K H., and Li, S F. Y (199 1) Separation of water and fat-soluble vttamins by mtcellar electrokmetic chromatography J Chromatogr 547,419428 77. FuJlwara, S , Iwase, S., and Honda, S. (1988) Analysts of water-soluble vttamins by mtcellar electrokmettc capillary chromatography J Chromatogr 447, 133-140 78 Ntsht, H., Tsumagart, N , Kaktmoto, T , and Terabe, S (1989) Separation of water-soluble vitamins by mtcellar electrokmettc chromatography J Chromatogr 465,33 1 79. Kenndler, E , Schwer, C., and Kaniansky, D (1990) Purtty control of riboflavm5’-phosphate (vttamm B, phosphate) by capillary zone electrophorests J Chromatogr

508,203

80. Kobayasht, S , Ueda, T , and Ktkumoto, M. (1989) Photodtode array detectton m htgh-performance captllary electrophoresis. J Chromatogr 480, 179-l 84 81. Thormann, W , Moltem, S., Caslavska, J , and Schmutz, A (1994) Clinical and forenstc applications of capillary electrophorests. Electrophoresis 15,3-l 2 82. Xu, Y. (1993) Capillary electrophoresis Anal Chem 65,425R-433R 83 Deyl, Z , Tagltaro, F , and Mtksik, I. (1994) Btomedtcal applications of captllary electrophorests. J Chromatogr 656,3-27 84. Thormann, W , Lienhard, S , and Wernly, P. (1993) Strategies for the momtormg of drugs in body fluids by micellar electrokmettc capillary chromatography J Chromatogr.

636,137-148

85 Tagltaro, F , Moretto, S , Valentmi, R , Gambaro, G , Anatoh, C., Moffa, M , and Tato, L (1994) Captllary zone electrophorests determination of phenylalanine m serum-a raptd, mexpenstve and simple method for the dtagnosis of phenylketonurta. Electrophoreszs 15,94-97 86. Lt, S., Frted, K., Warner, I W., and Lloyd, D. K. (1993) Determination of dextromethorpan and dextrorphan in urine by capillary zone electrophorests, apphcatton to the determmation of debrtsoqum-oxidation metabolic phenotype Chromatographta

35,216222.

87 Chen, F. T. A and Sternberg, J C. (1994) Charactertsation of protems by captllary electrophoresis m fused stltca columns-revtews on serum-proteins analysts and apphcatton to immunoassays. Electrophoresls 15, 13-2 1. 88. Guzman, N. A , Moschera, J., Iqbal, K., and Mahck, A. N. (1992) Effect of buffer constituents on the determmatton of therapeuttc proteins by capillary electrophoresis. J Chromatogr 608, 197-204.

Additional

Application

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89 Shihabi, Z. K. (1993) Serum phenobarbital assay by captllary electrophoresis J. Llqurd Chromatogr l&205!?-2068 90. Schmutz, A. and Thormann, W. (1993) Determinatton of phenobarbital, ethosuximide, and primrdone m human serum by mleellar electrokmettc capillary chromatography with direct sample inJection Therapeutic Drug Monztorzng, 15, 310-316 91 Caslavska, J., Ltenhard, S., and Thormann, W. (1993) Comparatrve use of three electrokinettc capillary methods for the determmatton of drugs m body flutds Prospects for rapid determmatton of mtoxications. J Chromatogr. 638, 335-342 92 Miyake, M , Shrbukawa, A , and Nagawaka, T (1991) Simultaneous determmatron of creatinine and uric acid m human plasma and urine by mrcellar electrokinetrc chromatography. JHRCC 14, 18 l-l 85 93. Kuhr, W G (1990) Capillary electrophoresis. Anal Chem 62,403R-4 11 R 94 Yik, Y F and Lr, S. F. Y. (1992) Captllary electrophorests with electrochemical detection TRAC l&325-332 95. Avdalovtc, N , Pohl, C A , Rocklm, R D , and Stillain, J. R (1993) Determmation of cations and anions by capillary electrophorests combined wrth suppressed conductivity detectron. Anal. Chem. 65, 1470-1475 96 Kobayashi, S , Ueda, T., and Krkumoto, M. (1989) Photodtode array detection m high-performance captllary electrophoresis. J Chromatogr 480, 179 97. Beck, W., Van Hoek, R., and Engelhardt, H. (1993) Applicatton of a drode-array detector m capillary electrophorests. Eiectrophoresls 14, 540-546 98. Niessen, W. M A., TJachen, U. R , and Van der Greef, J (1993) Caprllary electrophoresis-mass spectrometry J Chromatogr 636, 3-l 9. 99 Deterding, L. J., Moseley, M A., Tomer, K. B., and Jorgenson, J W (1991) Nanoscale separations combrned with tandem mass spectrometry. J Chromatogr 554,73-82. 100. Remhold, N J., Tmke, A. P , Tjaden, U. R., Niessen, W. M A, and Van der Greef, J. (1992) Captllary isotachophoretic analyte focussing for capillary electrophorests with mass spectrometrtc detection using electrospray tonizatton. J Chromatogr 627,263-27 1 101. Smtth, R. D., Wahl, J. H., Goodlett, D. R , and Hofstadler, S. A. (1993) Capillary electrophoresis I mass spectrometry. Anal. Chem 65,574A-584A 102. Johansson, I. M , Pavelka, R., and Henion, J. D. (1991) Determinatton of small drug molecules by capillary electrophorests-atmospheric pressure tomzatton mass spectrometry. J. Chromatogr 559,5 15-528 103 Kostamen, R., Lasonder, E , Bloemhoff, W., Vanveelen, P A., Welling, G W., and Brums, A. P. (1994) Charactertsation of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresrs/electrospray (tonspray) mass spectrometty and 252Cf plasma desorption mass spectrometry. Biol. Mass Spectrom 23,346352 104. Lamoree, M. H., Reinhold, N J., Tjaden, U. R., Niessen, W. M. A., and Van Der Greef, J. (1994) On-line tsotachophoresis, for loadabihty enhancement m capillary zone electrophorests/mass spectrometry of P-agonists. Blol. Mass Spectrom. 23,339-345.

342

Altria

105 Skoclr, E , Vmdevogel, J , and Sandra, P. (1994) Separation of 23 danyslated ammo acids by mlcellar electrokinetlc chromatography at low temperatures Chromatographza

39,7-10

106 Terabe, S., Ishlhama, H , Nlshl, H , Fukuyama, F , and Otsuka, K. (1991) Effect of urea addltlon m mlcellar electrokmetlc chromatography. J Chromatogr 545,359 107 Waldron, K C , Wu, S , Earle, C W , Harke, H R., and Dovlchl, N J (1990) Capillary zone electrophoresls separation and laser-based detection of both fluorescem thiohydantom and dlamethylammoazobenzene thiohydantom derivatives of amino acids. Electrophoresv 11,777-780. 108 Wu, S and Dovlchl, J N. (1992) Capillary zone electrophoresls separation and laser-induced fluorescence detection of zeptomole quantities of fluorescem thlohydantoin derivatives of ammo acids. Talanta 39, 173-l 78. 109 LIU, J , Hsleh, Y , Wlesler, D., and Novotny, M (1991) Design of 3-(4carboxybenzoyl)-2-qumolmecarboxaldehyde as a reagent for ultrasensltlve determination of primary ammes by capillary electrophoresls using laser fluorescence detectlon. Anal Chem 63,408-412. 110 Toulas, C and Hernadez, L (1993) Apphcatlons of a laser-induced fluorescence detector for capillary electrophoresls to measure attomolar and zeptomolar amounts of compounds. LC GC lo,47 l-476 111 Camillen, P , Dhanak, D , Druges, M , and Okafo, G. (1994) High sensmvlty detection of ammo acids usmg a new fluorogemc probe. Anal Proc. 31,99-102 112 Ong, C P , Ng, C L , Lee, H K , and LI, S F Y (1991) Separation of Dns-amino acids and vitamins by mlcellar electrokmetlc chromatography J Chromatogr. 559,537-545

113. Remhoud, N J , Tjaden, U. R , and Van der Greef, J. (1994) Automated on-caplllary Isotachophoretlc reactlon cell for fluorescence derlvatlsatlon of small sample volumes at low concentrations followed by capillary zone electrophoresls .I Chromatogr

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114. Nickerson, B and Jorgenson, J W. (1988) High speed capillary zone electrophoresls with laser induced fluorescence detection JHRCC 11,533,534. 115. Kuhr, W G. and Yeung, E. S (1988) Indirect fluorescence detection of native ammo acids in capillary electrophoresls Anal Chem. 60, 1832-1834 116 Jones, H. K, and Ballou, N. E (1990) Separations ofchemlcally different particles by capillary electrophoresls Anal Chem. 62,2484-2490 117. Amankwa, L. N., Scholl, J , and Kuhr, W. G. (1992) Characterlsatlon of the ollgomerit dispersion of poly(oyxalkylene)diamine polymers by precolumn derlvatlsatlon and capillary zone electrophoresls with fluorescence detectlon. Anal Chem. 62,2189-2193

118. McCormick, R M. (199 1) Characterlsatlon of slhca sols using capillary zone electrophoresls. J Llquld Chromatogr 14,939-952 119. Ebersole, R. C. and McCormick, R. M (1994) Separation and isolation of viable bacteria by capillary zone electrophoresls &o/technology 11, 1278. 120. Burkmshaw, S M , Hinks, D., and Lewis, D M (1993) Capillary zone electrophoresis in the analysis of dyes and other compounds m the dye industry and dyeusing industries J Chromatogr 640,4 13-4 17

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12 1. Hmks, D and Lewrs, D M (1993) Capillary electrophoresis of dyes Chromatogr Anal. AugJSept., 9-11. 122 Yang, Q., Smeyers-Verbeke, J , Wu, W , Knots, M. S., and Mussart, D. L. (1994) Sunultaneous separatton of ammotuum and alkali, alkahne earth and transrtion metal tons m aqueous-orgamc media by captllary ton analysis. J. Chromatogr. 688,339-349 123 Desbene, P L., Morm, C J., Desbene Monvemay, A. M , and Groult, R. S (1995) Utihsation of fluorescein sodium salt m laser-induced indirect fluorimetric detectton of tons separated by capdlary zone electrophorests. J Chrovnatogr 689, 135-148. 124. Roder, A and Bachmann, K (1995) Simultaneous determmatton of organic and inorganic anions m the sub-pmol range m ram water by capillary electrophoresis J Chromatogr 689,305-311 125. Dabek-Zlotorynska, E. and Dlouhy, J F. (1994) Captllary zone electrophoresls wtth indnect UV detectton of orgamc anions using 2,6-napthalene dlcarboxylic acid J Chromatogr. 685, 145-153 126. Oefner, P. J (1995) Surface-charge reversed capillary zone electrophoresis of inorganic and organic amens. Electrophoresis 16,46-56. 127 Timerbaev, A. R and Semenova, 0. P (1995) Theoretical esttmatton of captllary zone electrophoresis behavtour of metal complexes using multivartate regression analysts. J Chromatogr A 690, 141-148 128. Oefner, P., Chtesa, C., Bonn, G., and Horvath, C (1994) Developments in capillary electrophorests of carbohydrates J Cap Electrophor 1, 5-26 129. Mechref, Y., Ostrander, G K , and Elrassi, Z. (1995) Capillary electrophorests of carboxylated carbohydrates. 1. Selecttve precolumn derivattzatron of gangliosrdes with UV absorbing and fluorescent tags J Chromatogr 695, 83-95 130 Ye, J and Baldwin, R. P. (1994) Determinatton of carbohydrates, sugar acids and aldrtols by capillary electrophorests and electrochemtcal detection at a copper electrode J Chromatogr 687, 141-148 131 Klockow, A, Paulus, A, Ftguerredo, V., Amado, R., and Wtdmer, H. M (1994) Determinatton of carbohydrates m fruit juices by capillary electrophoresrs and hrgh-performance hquid chromatography J. Chromatogr 680, 187-200 132. Sht, H., Ma, Y. F., Humphrey, J. H., and Craft, N E (1995) Determmatton of vttamm A m dned blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection. J Chromatogr. B. 665, 89-96. 133. Zhang, Z. Y , Fasco, M. J., and Kammsky, L. S (1995) Determmatlon of theophylline and its metabohtes in rat liver mlcrosomes and human urine by caprllary electrophoresrs. J. Chromatogr B. 665,201-208. 134. Chicharro, M , Zapardrel, A., BernIeJo, E , Perex-Lopex, J A., and Hernandez, L. (1995) Direct determmatron of ephedrme alkaloids and epinephrme in human urine by capillary zone electrophorests. J. Lzq Chromatogr 18, 1363-l 38 1. 135 Bogan, D. P., Deasy, B., Okennedy, R., Smyth, M. R., and Fuhr, U (1995) Determination of free and total 7-hydroxycoumarin content m urine and serum by capillary electrophoresis J Chromatogr B 663,37 l-378 136. Lloyd, D. K. and Watzig, H. (1995) Sodium dodecyl sulphate IS an effective between-run rinse for capillary electrophoresrs of samples m biologtcal matrices. J. Chromatogr B 663,400-405.

Al tria 137 Zhang, C.-X and Thormann, W (1994) Determmatlon of drug levels m human serum by mlcellar electrokmetlc capillary chromatography with direct sample InJectIon using different quantttatlon strategies. J Cup. Electrophor 1,208-2 18 138 Amankwa, L N , Harder, K , Jink, F , and Aebersold, R (1995) High-sensltlvlty determmatlon of tyrosme-phosphorylated peptldesby on-line enzyme reactor and electrospray lomsatlon massspectrometry Pvot Scz 4, 113-125 139 Janning, P , Schrader, W , and Lmscheld, M (1994) A new massspectrometrx approach to detect modlficatlons m DNA. Rapzd Comm Mass Spectrom. 8,1035-1040