CEC Separation

The effect of pore size on CEC separation was also studied in detail [70-75]. Figure 9 shows the van Deemter plots for a series of 7-pm ODS particles with pore size ranging from 10 to 400 nm. The best efficiency achieved with the large pore packing led to a conclusion that intraparticle flow contributes to the mass transfer in a way similar to that of perfusion chromatography and considerably improves column efficiency. The effect of pore size is also involved in the CEC separations of synthetic polymers in size-exclusion mode [76]. 

Another way to improve the performance of open-tubular columns was suggested by Sawada and Jinno [83]. They first vinylized the inner surface of a 25 pm i.d. capillary and then performed in situ copolymerization of f-butylacryl-amide and 2-acrylamido-2-methyl-l-propanesulfonic acid (AMPS) to create a layer of polymeric stationary phase. This process does not currently allow good control over the homogeneity of the layer and the column efficiencies achieved in CEC separations of hydrocarbons were relatively low. These authors also recently thoroughly reviewed all the aspects of the open tubular CEC technologies [84]. 

Fig. 16. CEC separation of naphthalene (1), fluorene (2), phenanthrene (3), anthracene (4), pyrene (5),triphenylene (6),andbenzo(a)pyrene (7) using capillary filled with CIO alkyl substituted polyallylamine. (Reprinted with permission from [86]. Copyright 1997 Elsevier). Conditions capillary 50 pm i.d., 48 cm total length, 33 cm active length, field strength 400 V/cm, carrier concentration 20 mg/ml, mobile phase 60 40 methanol-20 mmol/1 borate buffer pH=9.3...

Columns filled with polymer solutions are extremely simple to prepare, and the packing can easily be replaced as often as desired. These characteristics make the pseudostationary phases excellent candidates for use in routine CEC separations such as quality control applications where analysis and sample profiles do not change much. However, several limitations constrain their widespread use. For example, the sample capacity is typically very low, pushing typical detection methods close to their sensitivity limits. Additionally, the migration of the pseudostationary phase itself may represent a serious problem, e. g., for separations utilizing mass spectrometric detection. The resolution improves with the concentration of the pseudostationary phase. However, the relatively low solubility of current amphiphilic polymers does not enable finding the ultimate resolution limits of these separation media [88]. 

Fig. 27. CEC separation of a neutral test mixture at in mobile phases with different pH values. (Reprinted with permission from [ 148]. Copyright 2000 Elsevier). Conditions capillary column 100 pm i.d., total length 33.5 cm, active length 25 cm packed with 3 pm Waters Spherisorb ODS I, mobile phase (A) 4 1 acetonitrile-25 mmol/1 TRIS pH = 8 (B) 4 1 acetonitrile-25 mmol/1 phosphate, 0.2 % hexylamine pH = 2.5, voltage 25 kV, temperature 20 °C. Peaks thiourea (1), di-methylphthalate (2), diethylphthalate (3), biphenyl (4), o-terphenyl (5)... 

Such columns can be used for the CEC separation of small neutral compounds. The problem with this type of open-tubular column, however, is the low efficiency obtained due to the small diffusion coefficients of the analytes in the polymeric stationary phase, and the heterogeneous film structure caused by Rayleigh instability. 

CEC separation of barbiturates Determination of barbiturates in human serum... 

Determination of amino acids in the analysis of dialyzed rat cerebrospinal fluid Chiral CEC separations of amino acids... 

Affinity-based CEC separations for the analysis of mannose-binding proteins... 

Summary of Organic Monolithic Polymer Systems That Have Been Introduced in Literature Listed Together with Their Mode of Polymerization, Porogenic Solvent, and Their Key Application in HPLC and CEC Separation... 

Fig. 3. IEC/CEC separation of anions and cations. Column TSK-Gel OA-Pak A (300 X 7.8 mm, 5 jam) eluent 5 mM malic acid-methanol (95 5) flow rate 1.2 ml/min sample volume 25 jal detection conductivity. Peaks (1) sulfate, (2) chloride, (3) nitrate, (4) fluoride, (5) sodium, (6) ammonium, (7) potassium, (8) magnesium, (9) calcium. Reprinted with permission from [19].

Much has been written about the usefulness of MIPs for chiral HPLC or chiral CEC separations. As long as one has an expert in chiral separation and can afford the expensive chiral HPLC columns which are commercially available, there seems to be little incentive to use MIPs for this purpose [25]. Chiral separations are today often required in the pharmaceutical industry, where the expertise and the financial resources are available to solve most problems. In the future, however, there may be more interest in chiral separation by other scientists, e.g., biochemists, and this could contribute to a wider use of MIPs in analytical chiral separations. 

The problem that reduction of the plate heights will not considerably improve the peak shapes on MIP phases has perhaps not been recognized immediately when MIP CEC was developed. A recent (2006) review by Ch. Nilsson and S. Nilsson [13] concludes, however So far all verified imprint-based CEC separations showed peak tailing, due to polydispersity of the imprinted receptor sites, resulting in different affinity and poor mass transfer. Although we do not agree with the exact wording (because the isotherm may be nonlinear even if all sites are the same... 

A similar HD injection procedure was also developed for CEC separation of 3-FITC-labeled peptides (see Figure 4.19). This HD injection has resulted in less biased amounts of the faster moving components (see Figure 4.19b), as compared to the case when EK injection was used (see Figure 4.19a) [566]. 

Other than beads, porous polymer monoliths, which were photopolymerized in a COC chip, were used for solid-phase extraction. It is known that priming polymeric surfaces is not as simple as priming silica surfaces, which use a common surface primer agent, TMPM. Therefore, the grafting method as initiated by UV should be used to attach the polymer monoliths [588]. A similar strategy was used for sample pre-concentration of PAHs (e.g., pyrene). Pyrene (900 nM) was first concentrated by 400-fold in 24% ACN before switching to 56% ACN for CEC separation (see Figure 5.5) [148]. 

Electrochromatography was first demonstrated in the open-tubular microfluidic channel coated with octadecylsilane (ODS) as the chromatographic stationary phase. EOF was used as the pumping system within the 5.6-pm-deep and 66-pm-wide channels. Plate heights of 5.0—44.8 pm were achieved for three cou-marin dyes [112]. Open-channel CEC separation was achieved using a stationary phase of octadecyltrimethoxysilane [148], CEC separation of some PAHs has also been carried out on a Pyrex chip with a thin film of C8 stationary phase formed using a sol-gel process [338],... 

CEC separation has been achieved on a reverse-phase chromatography bed, which is a 200-pm bed packed with ODS-coated silica beads (see Figure 5.4 in... 

Chapter 5). Separation of a mixture of BODIPY and fluorescein was achieved in less than 20 s (see Figure 6.23). Five bioactive peptides (papain inhibitors, proctobin, opioid fragment 90-95, ileangiotensin HI, and angiotensin III) were also separated in this manner [639]. A similar method has been used to separate two coumarin dyes or to separate Alexafluor-labeled angiotensin from the excess dye [587,640]. CEC separation of four FITC-labeled synthetic peptides was... 

To increase the surface area of the stationary phase for CEC separation, a collocated monolithic support structure (COMOSS) was constructed in a Si chip. A polystyrene-sulfonic acid stationary phase was then immobilized [349]. Design of the COMOSS required that the combined cross sectional area at the column head to be the same at any point in the inlet distributor [644]. A study for the reduction of band broadening in COMOSS was also reported [645]. 

The COMOSS has also been fabricated on a PDMS chip for CEC separation of FITC-labeled peptides (Figure 6.25). However, in the CEC separation of a mixture of rhodamine and fluorescein, a broad rhodamine peak was obtained, but fluorescein did not have this problem. This was possibly because the neutral rhodamine had diffused into the PDMS substrate, as illustrated in the fluorescent image in Figure 6.26 [360]. In another report, CEC separation of a peptide mixture was performed on a PDMS chip after cerium(IV)-catalyzed polymerization of the stationary phase within the channels [646]. 

FIGURE 6.24 (a) Isocratic, (b) step gradient, and (c) linear gradient used in the CEC separations of (1) anthracene, (2) pyrene, (3) 1,2-benzofluorene, and (4) benzo[a]pyrene. The injection time was 20 s. The concentrations of analytes 1 1 were 2.8, 0.9, 5.8, and 5.0 M, respectively. The dashed lines show the gradient profiles as seen at the detector [148]. Reprinted with permission from the American Chemical Society. 

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