Development of methods to improve sensitivity and portability of capillary electrophoresis for the analysis of stabilizers and drugs 2

Chapter Introduction 1.1 Basic Theory of Capillary Electrophoresis Analytical techniques, when based on the migration of electrically charged particles of ions in solution in an applied electric field, can be classified as electrophoresis. Capillary electrophoresis (CE), also designated as high performance capillary electrophoresis (HPCE), normally carried out in fused silica capillaries, is based on the same basic theory as conventional electrophoresis. As CE is a liquid-phase separation technique, it has close resemblance to high performance liquid chromatography (HPLC), particularly ion chromatography and reversed-phase HPLC although there are significant differences. The comparison is in terms of the following aspects: 1, The basic separation principle is different. CE is mainly based on different migration speeds of charged particles under an electrical field to achieve the separation of components, which are the ionic differences between the analytes. While reversed-phase HPLC, as well as gas chromatography (GC), is mainly based on polarity differences of the analytes. 2, In contrast to HPLC where the mobile phase is constantly being pumped into the system, in CE, the ideal experimental conditions not require a physical flow of the buffer across the column, since the driving force for the actual migration of species in the column in CE are their charges, the applied potential and EOF. 3, Since the capillary applied in CE is normally empty (except Capillary Gel Electrophoresis (CGE) or Capillary Electrochromatography (CEC)), while the HPLC column is normally filled with particles so the longitudinal molecular diffusion (Eddy diffusion) and mass transfer restrictions encountered in liquid chromatography (LC) are not relevant in CE. As a result, the separation efficiency of CE is significantly better than with HPLC. 4, CE columns, whose internal diameters (ID) normally range from 25μm to 150μm, are much smaller than HPLC columns, whose IDs are normally 1mm to 4.5mm. This difference mainly results in two advantages and one disadvantage. The first advantage is that the solvent/buffer consumption of CE is very much lower than the amount of organic mobile phase usually required for HPLC. Consequently, it is not only more economical to use CE than HPLC for separations but also more environmentally friendly, as aqueous-based buffers instead of organic solvents in HPLC alleviates the problem of waste disposal. Therefore, from a long term perspective, the use of CE seems both economically and environmentally desirable. The second advantage is the smaller amount of sample loading in CE, which is especially useful in bioanalysis because a lot of bio-samples are very expensive or precious. However, the small column also results in much shorter detection pathlength in CE, which will strongly decrease the concentration sensitivity of CE technique. So the CE sensitivity is normally 10 to 20 times less than that of HPLC for absorbance detection while all the other conditions are the same. The basic concept of CE is that an electrically charged species moves in a certain speed under the influence of an electric field. Compounds are separated due to their different migration speeds. The velocity of an ion is given by the following equation: ν = μE = Leff / tm [unit: m/s] (1.1) where, ν: the velocity of the component; μ: electrophoretic mobility of the component; E: the electric filed strength; Leff: the effect length of capillary, the capillary length from inlet to the detector tm: migration time of the component After steady state is reached, the ions move with a constant velocity ν. The velocity ν is proportional to the applied electric field as well as the electrophoretic mobility μ, which is a characteristic property of a given ion in a given medium and at a given temperature. So the electrophoretic mobility μ of the ions relates to ν and E. The velocity of an ion is determined by dividing the traversed capillary length Leff, by the migration time tm of the peak. The electrophoretic mobility μ can also be expressed as μ= q / 6πηr = ν/E [unit: cm2/(V-s)] (1.2) where; q: ion charge; η: solution viscosity [Pa-s] r: ion radius [cm] So the viscous drag of the solvent and the charge and size of the solute thus control the migration of a species in an applied electric field. The separation efficiency, normally expressed as the number of theoretical plates (N), can be defined using the following equation: N = ( L/ σ)2 (1.3) Where: L: distance from injection point to detection point; σ: total spatial variance of the concentration profile of the zone. If the peaks acquired are symmetric and have Gaussian profile, the theoretical plate number can also be calculated from the electropherogram using the following equation: N = 5.54 (t/w1/2)2 (1.4) Where; t: migration time; w1/2: temporal peak width at half of the peak height Compared with HPLC, the efficiency of CE is normally higher mainly due to the plug-shaped flow profile [1]. However there is still zone broadening in CE technique. The most important causes of zone broadening in CE are the following: 1. Longitudinal diffusion: It corresponds to the theoretical limit, increases directly with analysis time and the diffusion coefficients, and inversely with the molecular weight. 2. Thermal effects: The thermal effects lead to convection and to local changes in buffer viscosity. 3. Injection length: If the injection length is too big, then broad peak and poor resolution will result, though the detection limit may be increased. So it should be smaller than the zone generated by diffusion, normally less then 1% of the effective length of the capillary. 4. Wall adsorption of samples: It causes peak tailing and poorly reproducible migration times, and can be decreased by using coated capillary and other more complicated method. 5. Electrodispersion (Mobility difference): The electrodispersion causes “triangular” peak shapes 6. Difference in the liquid levels: This may cause hydrodynamic flow, that’s why one of the basic requirements of a CE instrument is to make the inlet and outlet vial at the same level. 1.2 Electroosmotic Flow (EOF) Electroosmotic flow (EOF), which causes the buffer solution to flow in an electric field, is very important in CE. It depends upon the distribution of charge in the proximity of the capillary surface. Generally, nearly all surfaces carry a charge, and in the case of quartz capillaries wall, there are negative charges from the dissociation of the silanol groups. In the solution these surface charges are counterbalanced by counterions, which means oppositely charged ions. As Figure 1.1 shows, in the double layer, due to the negative charges of the fused silica surface, the positive ions predominate in solution are arranged in a rigid and a diffuse layer. According to Stern’s theory, the potential built up on account of the charge distribution is divided into two regions: the rigid boundary layer with adsorbed ions and Stern boundary layer (diffuse boundary layer). A linear decrease in the potential in the region of the first layer and an exponential decrease in the rear layer were observed in experiments. The exponential decrease is responsible for the electroosmosis and is designated as the ζ–potential. Figure 1.1: Charge distribution at the surface of fused silica and formation of the ζ–potential Figure 1.2: Profile of the ζ–potential at the buffer/fused silica interface a: rigid boundary layer with adsorbed ions; b: Stern boundary layer (diffuse boundary layer); c: Electrolyte The migration velocity ν of the EOF can be described with following equation: νeo = ε E ζ / 4πη [unit: m/s] (1.5) Where; ε: the dielectric constant of electrolyte E: the applied field strength ζ: the ζ–potential (zeta-potential) η: the viscosity of the electrolyte νeo is also described by the equation νeo = u eo (V/L) [unit: m/s] (1.6) Where; u eo: electroosmotic mobility V: applied voltage; L: capillary length. As the electric field is normally applied parallel to the wall surface (see Figure 1.1), the field pulls the counterions of the mobile layer along its axis and so moves the entire liquid in the capillary along with it. The EOF is induced to move to the cathode in quartz capillaries with an enrichment of positive ions in the boundary layer. An extremely flat (piston-shaped) flow profile is produced as shown in Figure 1.3. Calculations have shown that typically for capillary electrophoresis liquid layers as little as 10nm from the quartz surface already move uniformly under experimental conditions [1]. As a result, this leads to substantially less band broadening than for hydrodynamic flow (see Figure 1.3). In capillaries with inner diameters of 25 μm to 100 μm the flow profile can be regarded as nearly ideally plug-shaped. On the other hand in hydrodynamic flow, the parabolic Hagen-Poiseuille flow profile appears. This round head shaped (parabolic) flow profile is strongly dependent upon the capillary radius and the flow velocity. The difference in flow profile results in different peak shapes as in Figure 1.3, which shows a comparison of both flow profiles in HPLC and CE as well as the peak shapes in both techniques. (A) Flow profile in HPLC (B) Flow profile in CE Figure 1.3: Representation of a pressure-generated flow profile (A) and an ideal plug flow profile (B). The EOF appears in nearly all electrophoretic separation methods because surface charges cannot be completely eliminated. As the EOF is normally from anode to cathode, the detector in CE is usually placed on the cathode side. All the cations, neutral components and anions move with the EOF as shown in Figure 1.4. But cations will move faster than the EOF as their velocity direction is the same as that of the EOF, so very rapid analysis times are therefore achieved with positively charged compounds. The neutral compounds move at the same speed as EOF, so they cannot be separated in Capillary Zone Electrophoresis (CZE). However for anions, though they migrate opposite to the direction of the EOF, are also transported to the detector (on the cathode side). Since their migration velocities are typically lower than the velocity of the EOF, they can still be detected in CZE. Therefore, under suitable conditions cations and anions can be separated from each other in a single analysis. Only the anions that migrate faster than the flow velocity of the EOF migrate into the anode compartment and escape detection. These ions can be detected by reversing the polarity, but then the cations and the slowly migrating anions escape detection. Figure 1.4: Separation in Capillary Zone Electrophoresis It is possible to detect cations and anions in the same run by controlling the EOF to elute more components of the sample. There are several frequently used methods to change the EOF, such as adjustment of pH value, varying electrolyte concentration of 10 experiments. Data acquisition and recording of electropherograms were accomplished with CE-PKS chromatography Station (CE Resources Pte Ltd, Singapore). 6.2.2 Chemicals and Solutions L-carnitine was a gift from Wako (Osaka, Japan). Citric acid and glacial acetic acid were purchased form Sigma (St. Louis, USA). Phosphoric acid was obtained from Fluka (Buchs, Switzerland). Boric acid was a product of Aldrich (Milwauke. USA). Di-ammonium hydrogen citrate was obtained from BDH (Poole, England). The carnitine real sample, Metabolism Increaser capsule (MIc), was manufactured by 21st Century (Arizona, USA). Deionized water used throughout the experiments was supplied by a Nanopure ultrapure water purification system (Barnstead, IA, USA). For CE experiments, unless otherwise mentioned, the separation buffer consisting of 1.0 mM ammonium citrate buffer (pH=3.8) was prepared daily and stored in a 4oC refrigerator. L-carnitine standard stock solution (1% w/w) was prepared by dissolving L-carnitine standard sample in pure water weekly and stored in a 4oC refrigerator. L-carnitine standard sample solutions for injection were made by diluting the stock solution to the required concentrations. Metabolism Increaser capsule, which is white color powder inside a packaged 145 capsule, was dissolved to make 1% (w/w) solution. After filtering with a 0.22 μm cellulose syringe filter, the stock solution was ready. These stock solutions were quite stable and were stored in a 4oC refrigerator. Before introducing into the capillary for electrophoresis, these stock solutions were diluted to the required concentration using deionized water. 6.2.3 Procedure for CE Experiment The new capillary was treated by flushing 100mM NaOH solution, pure water and running buffer for minutes sequentially. Between two consecutive runs the capillary was flushed with running buffer for min. The samples were hydrodynamically injected into the capillary under a positive pressure of 2.07 x 103 Pascal for 10 seconds. After sample introduction, 25 kV voltages were applied for separation. The CE experiments were carried out at room temperature of 25 oC. 6.3 Results and Discussion 6.3.1 Basic Consideration According to the structure of carnitine in Fig.6.1, carnitine has an amino group, which will render the molecules positively charged in low pH solutions. In C4D, suppressed conductivity of buffer allows lower LOD to be achieved since a suppressed system enlarges the difference between the conductometric signal of the analytes and the background of buffer electrolytes. Therefore, the acidic, low-conductivity buffers were chosen in our experiments as starting point for 146 optimizing the buffer conditions for the analysis of carnitine. 6.3.2 Buffer Optimization 6.3.2.1 Buffer System Figure 6.3. Optimization of buffer system: A) 1mM ammonium citrate (pH 3.8), B) 1mM acetic acid buffer (pH 4.0), C) 1mM borate buffer (pH 6.0) and D) 1mM phosphate buffer (pH 3.8). Peaks 1, and are identified as L-carnitine, EOF and impurity respectively. Conditions: injection, 2070 Pa x 10 s; Voltage, 25 kV. Sample: 40 ppm L-carnitine. Figure 6.3 shows the electrophoregram obtained for several different buffer systems. From the results, we can see that the ammonium citrate buffer system is the best system for this analysis, which is not only fastest, but also most sensitive. The possible reason is that the ammonium ion has small molecular mass and has a 147 faster mobility than that of carnitine, which is better for C4D detection of carnitine. The acetic acid system (Fig. 6.3 B) can also produce a smooth base line, but the peak is much broader and eluted slower, which means lower sensitivity and longer analysis time. No L-carnitine peak was observed for the borate buffer system (Fig. 6.3 C), which may be due to the fact that the phosphate buffer system (Fig. 6.3 D) exhibited large noise and broad peaks, which would lead to poor sensitivity for carnitine analysis. 6.3.2.2 Effect of pH Value Figure 6.4. Optimization of pH value: A) pH 3.2, B) pH 3.8 and C) pH 4.0 of 1mM ammonium citrate. Peaks and are identified as L-carnitine and EOF respectively. Conditions: injection, 2070 Pa x 10 s; Voltage, 25 kV. Sample: 40 ppm L-carnitine. 148 Separation buffers with various pH values were used to investigate the effect of pH. The results are shown in figure 6.4 and pH 3.8 was found to give the best results. Lower pH value (pH 3.2, Fig. 6.4 A) would lead to noisy base line as well as broad peaks. Though higher pH value (pH 4.0, Fig. 6.4 C) could also provide a smooth baseline, the peaks were less sharp and eluted later, which would not only lead to lower sensitivity but also longer analysis time. 6.3.2.3 Linearity, Repeatability and Limits of Detection Using the above optimum conditions, linearity and repeatability of the method were tested. To calculate the relative percentages of the main components of carnitine, corrected areas were used. The corrected areas were obtained by dividing the areas of the peak by their migration times. Figure 6.5. Linearity of carnitine standard sample. Conditions: separation buffer, mM ammonium citrate buffer (pH 3.8); injection, 2070 Pa x 10 s; Voltage, 25 kV. 149 Fig. 6.5 shows that the linear range of this method for carnitine analysis was from ppm to 1000 ppm (At concentrations higher than 1000 ppm, the peaks would be too broad and overlapping would occur, and hence 1000 ppm was chosen as the upper limit of the linear range.) with R2 values of 0.9993 (see Fig 6.6), which is much wider than that of other methods (e.g. ppm to 50 ppm ) [21], especially those method with low LOD up to several ppm levels. For the samples with concentrations higher than 1000 ppm, overloading would occur. The slope for carnitine was 0.0013, as shown in the calibration curve (Fig. 6.5). The LOD of this method was ppm based on S/N ratio ≥ and the LOQ of this method was 3.3 ppm based on S/N ratio ≥10, which is slightly higher than the best sensitivity reported to date, i.e. 0.5 ppm, obtained by the derivatization method [16]. Figure 6.6. Reproducibility of six runs of carnitine. Conditions: separation buffer, mM ammonium citrate buffer (pH 3.8); injection, 2070 Pa x 10 s; Voltage, 150 25 kV. Sample: 40 ppm L-carnitine. Peak 1, L-carnitine; Peak 2, EOF. Table 6.1. Repeatibility of CE-C4D method for analysis of carnitine standard sample Run The Same Day Differ -ent Day Migration Time/min Corr.Area/ x10-3mV Mean Value RSD, % (n=6) 4.309 4.23 1.34 55.28 55.02 Mean Value 1.40 RSD, % (n=3) 4.20 0.17 53.91 4.81 4.140 4.183 4.210 4.249 4.275 53.98 55.76 53.92 55.65 55.53 Day Day (n=6) Day (n=6) Day (n=3) Migration Time/min Corr.Area/ x10-3mV 4.20 4.23 4.18 55.95 55.02 50.77 Repeatability of the method was also investigated by repeated injection of the same sample into the capillary (Fig. 6.6 and Table 6.1). Fig. 6.6 shows six repetitive runs of carnitine analysis under the optimum conditions. The migration times increased from run to run 6, probably due to the slight adsorption of the sample onto the capillary wall, resulting in a decrease of the EOF. There was a base line jump, which could be the system signal, before the sample peaks in the experiments. However, it was well separated from the sample peaks and did not interfere with the quantitative analysis. It was found that the repeatability, in terms of corrected area and migration time, was satisfactory with RSD < 1.4% and < 1.34% respectively in the same day and RSD < 4.81% and < 0.17% on different days (see Table 6.1). 151 6.3.3 Determination of Real Sample Figure 6.7. Reproducibility of real sample analysis. A)~C) 100ppm MIc, D)~F) 100ppm MIc spiked with 50ppm standard L-carnitine; Conditions: separation buffer, mM ammonium citrate buffer (pH 3.8); injection, 2070 Pa x 10 s; Voltage, 25 kV. Peak 1, unknown; Peak 2, L-carnitine; Peak 3, EOF. Table 6.2. Real samples analysis results 100ppm MIc,s Run piked with50 ppm standard MigrationTime/min sample Corr.Area/ x10-3mV MigrationTime/min 100ppm MIc Corr.Area/ x10-3mV 5.1 184.4 5.012 102.1 4.865 189.3 4.923 102.3 4.897 186.4 4.873 98.43 Mean RSD,% Value (n=3) 4.95 2.10 186.70 1.08 4.94 1.16 100.94 1.76 Fig. 6.7 shows electropherograms of a real carnitine sample, Metabolism Increaser 152 capsule (MIc) and MIc spiked with standard L-carnitine. In Table 6.2, the mean values and % RSDs are shown, which are satisfactory (< 2.1%). Although just a simple dilution and filtering pretreatment was used, no major interference from other components in the real samples was observed except the small unknown peak (Fig 6.7 peak 1). The results of the real sample obtained by our method were quite close to the concentrations labeled on the bottles by the manufacturers. Because no derivatization is needed for detecting carnitine in these samples, our method is simpler then the current standard procedures and the total time per analysis is reduced to less than minutes. 6.4 Conclusion A new method was developed to determine native carnitine using capillary zone electrophoresis with capacitively coupled contactless conductivity detection. Using this method, the L-carnitine was baseline separated from the impurities and detected without derivatization. It has shown good sensitivity as well as very wide linear range from ppm to 1000 ppm, which is much wider than those of other methods previously reported. This method’s good repeatability, simplicity and rapidity could render itself a good alternative method to the current assays given by US Pharmacopoeia. The experiments were developed on a portable capillary electrophoresis system, which can also fulfill on-site analysis easily. 153 References [1] J.Bremer, J. Biol. Chem., 237, 1962, 3628 [2] A.L. 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A, 895, 2000, 309 [17] C. Vogt, S. Kiessig, J. Chromatogr. A, 745, 1996, 53 [18] T. Hirota, K. Minato, K. Ishii, N. Nishimura, T. Sato, J. Chromatogr. A, 673, 1994, 37 [19] C. Mardonesa, N. Viziolib, C. Carduccib, A. Riosa, M. ValcaÂrcel, Anal. Chim. Acta, 382, 1999, 23 [20] K. Heinig, J. Henion, J. Chromatogr. B, 735, 1999, 171 [21] V. Prokoratova, F. Kvasnicka, R. Sevcik, M. Voldrich, J. Chromatogr. A, 1081, 2005, 60 155 Concluding and Future Work Developments of methods to improve the sensitivity or to simplify the analysis are two important topics in CE techniques. In this thesis, the work are focused on developing more sensitive methods to determine UV active chemicals, as well as simpler methods to fulfill on-site analysis of non-UV active chemicals without any derivatization on portable capillary electrophoresis systems. With the high separation efficiency, high resolution, simplicity and portability, CE has been proven a powerful method for the analysis of these target samples: UV active stabilizers, which include some neutral stabilizers in pharmaceutical / cosmetic products and gunpowder stabilizers, as well as non-UV active drugs, which include antibiotics and carnitine. In chapter 1, an introduction to the principle and practice of capillary electrophoresis was given. Subsequently, the scope of the research carried out in this thesis was described. In chapter 2, a modified field amplification sample injection was proposed and evaluated for improving the detection limit of selected neutral stabilizers in micellar electrokinetic chromatography on the basis of using positively mono-charged cyclodextrin as carrier and 1-adamantanecarboxylate as displacer. The modified field amplification sample injection method further improved the detection limit compared with conventional field amplification sample injection without the use of the displacer 156 plug. The displacer plug reduced the length of the concentrated sample zone and increased the peak height by slowing down the forward movement of the neutral sample associated with β-CD-NH2 and the backward movement of the neutral sample partitioned in the micelles of SDS. Stability of the inclusion complexes formed between the carrier and the neutral sample was a key factor for concentration factor in both the field amplification sample injection and modified one. But compared with the field amplification sample injection, the further enhancement of the concentration factor in the modified field amplification sample injection method was mainly dependent on the relative stability of the displacer-carrier complex to the stability of the neutral solute-carrier complexes, i.e. K displacer-β-CD-NH2 / Ksolute-β-CD-NH2 There was an optimal length of the displacer plug for every solute. The more stable the solute-carrier complex, the longer the optimal displacer plug. The modified field amplification sample injection showed the detection limit being more than 10 times lower for the model compounds compared with that obtained with sample dissolved in the separation buffer in hydrodynamic injection mode. The method is potentially useful in expanding applications of the micellar electrokinetic chromatoghaphy especially for neutral compounds with are not easily preconcentrated by conventional stacking techniques. In chapter 3, a simple method was optimized, validated and applied to determine stabilizer components in gunpowder and propellant samples using MEKC with UV detection on a portable CE system. The method can be used to detect gunpowder real 157 samples within 10 minutes. It has shown better sensitivity than HPLC methods for determination of stabilizer components in gunpowder real samples. The linear range was from ppm to 60 ppm and the LOD was 0.5 ppm. The good repeatability and simplicity shown by this method could render it a good alternate for the current assays given by HPLC which not only needs big and expensive equipments but also consume large volumes of organic solvents. The most promising feature of the MEKC method is that it can be performed using a portable instrument as describe in this thesis, as a result fulfill the requirements for testing on field, which is especially useful to army and anti-terrorist applications. The results compiled from the trials shows that CE is a suitable and reliable technique for the separation and analysis of the concentrations of stabilizers in the gunpowder samples that were extracted from the rounds. The results are reproducible and the analysis time is relatively short (approximately 10mins). The stabilizer contents in the extracted samples were calculated to be within ±0.06% of the readings from HPLC. Furthermore, the CE technique features portability and convenience of on-the-spot surveillance providing quick answers to aid decision making in emergence situations. In chapter 4, a simple and fast method was developed to determine non-UV active compounds directly without derivatization. The usefulness of the method was demonstrated by detecting the major components in aminoglycoside antibiotic mixtures using a portable capillary electrophoresis system with potential gradient detection. The gentamicin components were separated into several peaks in 15 minutes, 158 and components of neomycin could be separated within minutes. This method showed better sensitivity than other CE methods for determining underivatized gentamicin and neomycin with large linear range. Because of its good repeatability and simplicity, this new method could be a good alternative for the current assays given by US pharmacopoeia and European Pharmacopoeia. In chapter 5, a further improved method was developed to determine gentamicin’s two stereoisomeric components (C2, C2a) as well as other major components (C1 and C1a) using the same portable CE-PGD system as in chapter 4. Chiral separation was achieved by using 0.2mM CTAB, 1mM vancomycin and 1mM ammonium citrate buffer (pH 3.5). Large linear range, good repeatability and simplicity of the method could also render this method a good alternative method to methods given by US and European Pharmacopias also. Furthermore, the method was developed on a portable capillary electrophoresis system and therefore can be potentially useful for field detection. In chapter 6, a new method was developed to determine native carnitine using capillary zone electrophoresis with capacitively coupled contactless conductivity detection. Using this method, the L-carnitine was baseline separated from impurities and detected without derivatization. It could reach lower LOD levels than previous methods, as well as very wide linear range from ppm to 1000 ppm, which is much wider than those obtained by other methods. The good repeatability, simplicity and rapidity of this 159 method could render itself a good alternative method for the current assays given by US Pharmacopoeia. The experiments were also developed on a portable capillary electrophoresis system with C4D and hence could also be useful for field testing. To extend the usefulness of the methods developed, several additional experiments may be considered. The modified field amplification sample injection method can be extended to the analysis of other type of neutral samples. A small portable UV detector as well as a small portable CE-LIF system will widen the scope of on-site analysis by CE. A portable UV detector which will not only be easier for carrying around but will also require less electrical power so that prolonged on-site analysis can be performed will have greater potential in the determination of more complicated analytes and large number of samples. The portable CE-PGD and CE-C4D systems and the methods developed can be applied to the analysis of other drugs which lack UV absorption, such as kanamycin and streptomycin. Chiral separations, e.g. of D, L-carnitine, which may be useful in quality control in pharmaceutical manufacturing processes, may also be investigated on the portable CE system. 160 [...]... just ranges from 2 to 20 nl To increase the sensitivity, two approaches, either to increase the amount of analyte added to the capillary or to improve the sensitivity of the detector, may be applied However, due to the characteristics of the CE technique, both of these two approaches are limited For example, as the volumes of narrow-bore capillaries are very small, the introduction of a large sample... Δp: the pressure difference; r: the inner diameter of the capillary; 20 t: the injection time; η: the viscosity of the buffer; L: the total length of the capillary In the case of all hydrodynamic sample introductions, it is necessary that the material in the capillary is free to flow So it is not suitable for viscous solutions, which is the shortcoming of this injection method Normally, 1% of the total... total capillary length of sample solution is injected With electrokinetic loading, the inlet end of the capillary is immersed in the sample, the outlet in the separation buffer and a low voltage (1-10kv) is applied for durations of less then 1 min depending on the capillary length and ID The quantity, Q, of a component of the sample injected to the capillary can be represented by Q = (μep + μeo) π r2 Ui... by the ID of the column Since the signal of an absorbance detector is proportional to the optical path length, much less sensitivity is achieved in CE detection due to the small ID of the capillary then in HPLC detection Several specially designed cells, such as “bubble” cell, “Z” cell and rectangular capillaries, have been applied to increase the optical path in order to improve the sensitivity Photodiode... lead to unacceptably broad peaks and poor resolution The sensitivity of a detector is also too hard or expensive to be improved A more practical and moderate way to address this concern would be to use on-line concentration techniques [7-9], which is done by manipulating the composition of the sample and background solutions together with sample injection procedures without modification of the 22 instrumentation... from the capillary in order to preserve separation efficiency The EOF pump is used for this purpose The direction of pumping is always opposite that of the electrophoretic movement of the charged solutes The velocity of pumping should be lower than the electrophoretic velocity of the charged solutes 25 1.4 .2. 2 Field Amplified Sample Injection (FASI) Figure 1. 12 Field amplified sample stacking model of. .. another kind of stacking method in CE, is always performed with the sample zone between the BGE of higher and lower electrophoretic mobilities The former BGE is the leading electrolyte and the rear is the terminating electrolyte Typically, either cations or anions can be separated in one run with some exceptions [24 , 25 ] For cation analysis, the leading buffer is positioned at the cathodic end of the. .. adsorbed on the surface silanol groups A double layer of the detergent is formed, with the positive charges directed toward the electrolyte (The formation of a double layer is shown schematically in Figure 1.6.) Another kind of additive which can help to attain reversal of the EOF is polyamines, such as spermine With such coated capillaries, together with reversal of the field, separation of slowly and rapidly.. .the buffer and introducing buffer additives The buffer concentration and the pH value represent the most important parameters for optimizing separation Variation in the buffer concentration presents one of the simplest and most effective means of influencing the EOF of the separation system Normally the EOF increases with decreasing buffer concentration and enables analysis of highly negatively... distribution of analytes between two pseudophases Figure 1.7 Scheme of MEKC mode The separation of neutral compounds is in principle impossible in free solution CZE, because of the lack of self-electrophretic mobilities of neutral analytes and thus the only driving force for neutral analyutes is the EOF, which is equal for all neutral 14 components of a sample On the other hand, in MEKC, the neutral . flow of the buffer across the column, since the driving force for the actual migration of species in the column in CE are their charges, the applied potential and EOF. 3, Since the capillary. though they migrate opposite to the direction of the EOF, are also transported to the detector (on the cathode side). Since their migration velocities are typically lower than the velocity of the. of the solvent and the charge and size of the solute thus control the migration of a species in an applied electric field. The separation efficiency, normally expressed as the number of theoretical