High performance capillary electrophoresis - T.E.A.M.
High performance capillary electrophoresis - T.E.A.M. High performance capillary electrophoresis - T.E.A.M.
Modes Figure 29 CZE of reversed phase HPLC collected peptides Conditions: 30 mM, 17 mM phosphate, pH 8.2, V = 25 kV, i = 36 mA, l = 70 cm, L = 78.5 cm, id = 50 mm with 3X extended pathlength detection cell, l = 200 nm mAU 16 14 12 10 8 6 4 2 Fraction 15 11 13 15 17 19 Time [min] mAU 16 14 12 10 8 6 4 2 Fraction 18 10 12 14 16 18 Time [min] mAU 62 60 56 52 [d-tyr] neurotensin Neurotensin Under optimized conditions, the high efficiency is sufficient to resolve small differences in solute structure. As shown in figure 30, two 8-amino acid long peptides which differ only by the substitution of D-tyrosine for L-tyrosine can be resolved. This example illustrates the effect of conformational differences on mobility, since CZE cannot separate isomers without chiral additives. 48 44 40 6 7 Time [min] Figure 30 CZE separation of neurotensin isomers Neurotensin amino sequence: pGlu-Leu-Try-Glu- Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-and pGlu- Leu-Try-Glu-Asn-Lys-Pro-Arg-Arg-Pro-D-Tyr-Ile- Leu. Conditions: 30 mM tetraborate, pH 8.3, V = 18 kV, i = 42 mA, l = 50 cm, L = 65 cm, id = 50 mm with 3X extended pathlength detection cell, l = 200 nm, peptide concentration 2.5 × 10 -5 M each Determination of drugs and drug metabolites has also been successful by CZE. Figure 31a shows the analysis of Cefixime (CX), an oral cephalosporin antibiotic, and its metabolites. The strong hydrophilicity of these metabolites makes extraction from biological fluids difficult. For the same reason, separation by reversed phase liquid chromatography is insufficient, even with ion-pairing agents. Identification of the drug in human urine is shown in figure 31b. Quantitative analysis within about 3 % RSD was obtained. An example of the use of CZE for environmental analysis is shown in figure 32. In this study, conditions were optimized to separate aromatic sulfonic acids and related compounds. Here, 4-chlorobenzesulfonate is identified as a leachate at an environmental clean-up site in the U.S. Peak identification was confirmed by mass spectrometry. Along with 60
Modes CA a) liquid chromatography, CE was suggested to hold great potential for the separation and characterization of polar, non-volatile environmental samples. CX 0 5 10 15 20 25 M CX M 3 4 CA M 2 M 5 M 1 b) CZE has also been used for the separation of inorganic ions and organic acids, which has been traditionally performed by ion chromatography (figure 33). Indirect UV detection is necessary due to the lack of chromophores in these species. Indirect detection is performed using a highly absorbing species such as chromate or imidazole in the running buffer and monitoring at the absorbance maximum of that species (for example, 254 nm for chromate). Solute ions displace the chromate ions and a decrease in absorbance occurs when the zones pass the detector region. In order to decrease the analysis time for the cations shown here, a cationic surfactant (CTAB) was added to the running buffer to reverse the EOF toward the cathode. Due to the high mobility of small ions, however, the EOF is not strong enough to carry ions migrating in the opposite direction and thus anions cannot be analyzed simultaneously. In these small ion determinations, peak shapes are often skewed due to the conductivity differences between the running buffer and the ions. 0 10 20 30 40 50 Migration time [min] Figure 31 a) CZE of Cefixime (CX) and its metabolites. b) Analysis of human urine spike with CX and cinnamic acid (CA) 14 Conditions: 50 mM phosphate, pH 6.8, V = 15 kV, l = 50 cm, L = 72 cm, id = 50 mm, l = 280 nm Absorbance 0.0400 0.0200 4-chlorobenzenesulfonate Figure 32 Analysis of environmentally hazardous leachates by CZE 15 Conditions: 50 mM borate-boric acid, pH 8.3, V = 30 kV, i = 33 mA, l = 50 cm, L = 75 cm, id = 50 mm, l = 280 nm 0.0000 0 10 20 22.25 Time [min] 61
- Page 10 and 11: Scope The purpose of this book is t
- Page 12 and 13: Introduction 1.1 High performance c
- Page 14 and 15: Introduction sis, methods for on-ca
- Page 16 and 17: Principles 2.1 Historical backgroun
- Page 18 and 19: Principles that ion. The mobility i
- Page 20 and 21: Principles the exact pI of fused si
- Page 22 and 23: Principles µ EOF × 10 -4 (cm 2 /
- Page 24 and 25: Principles For the analysis of smal
- Page 26 and 27: Principles µ EOF ( × 10 -4 cm 2 /
- Page 28 and 29: Principles Total length Effective l
- Page 30 and 31: Principles Note that equation (15)
- Page 32 and 33: Principles determined by the capill
- Page 34 and 35: Principles Current (uA) 300 250 200
- Page 36 and 37: Principles The contribution of inje
- Page 38 and 39: Principles k' H N H, µm 0.001 0.58
- Page 40 and 41: Principles Figure 19 Electrodispers
- Page 42 and 43: Principles rapidly eluting ions, th
- Page 44 and 45: Principles 44
- Page 46 and 47: Modes Mode Capillary zone electroph
- Page 48 and 49: Modes 3.1.1 Selectivity and the use
- Page 50 and 51: Modes Name pK a Phosphate 2.12 (pK
- Page 52 and 53: Modes EOF No flow Figure 22 Elimina
- Page 54 and 55: Modes Absorbance 214 nm 0.05 0.04 0
- Page 56 and 57: Modes Type Comment Silylation coupl
- Page 58 and 59: Modes Type Result Comment Extremes
- Page 62 and 63: Modes Figure 33 Ion analysis of fer
- Page 64 and 65: Modes The separation mechanism of n
- Page 66 and 67: Modes the stationary phase in LC. S
- Page 68 and 69: Modes Amplitude 2 a) with a migrati
- Page 70 and 71: Modes CGE t = 0 t > 0 Polymer matri
- Page 72 and 73: Modes Crosslinked polyacrylamide, a
- Page 74 and 75: Modes a) ds 500 base pairs This sam
- Page 76 and 77: Modes and resolution with respect t
- Page 78 and 79: Modes 3.5 Capillary isotachophoresi
- Page 80 and 81: Modes 80
- Page 82 and 83: Instrumentation/Operation Diode-arr
- Page 84 and 85: Instrumentation/Operation Pressure
- Page 86 and 87: Instrumentation/Operation If sensit
- Page 88 and 89: Instrumentation/Operation Despite q
- Page 90 and 91: Instrumentation/Operation T, ˚C 10
- Page 92 and 93: Instrumentation/Operation 4.2.1.1 C
- Page 94 and 95: Instrumentation/Operation However,
- Page 96 and 97: Instrumentation/Operation Calculati
- Page 98 and 99: Instrumentation/Operation Method Ma
- Page 100 and 101: Instrumentation/Operation 4.3.3 Lin
- Page 102 and 103: Instrumentation/Operation Area (arb
- Page 104 and 105: Instrumentation/Operation 4.3.6 Ext
- Page 106 and 107: Instrumentation/Operation light int
- Page 108 and 109: Instrumentation/Operation 4.3.7.2 Q
Modes<br />
Figure 29<br />
CZE of reversed phase HPLC collected<br />
peptides<br />
Conditions: 30 mM, 17 mM phosphate, pH 8.2,<br />
V = 25 kV, i = 36 mA, l = 70 cm,<br />
L = 78.5 cm, id = 50 mm with 3X<br />
extended pathlength detection<br />
cell, l = 200 nm<br />
mAU<br />
16<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
Fraction 15<br />
11 13 15 17 19<br />
Time [min]<br />
mAU<br />
16<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
Fraction 18<br />
10 12 14 16 18<br />
Time [min]<br />
mAU<br />
62<br />
60<br />
56<br />
52<br />
[d-tyr] neurotensin<br />
Neurotensin<br />
Under optimized conditions, the high efficiency is sufficient<br />
to resolve small differences in solute structure. As shown<br />
in figure 30, two 8-amino acid long peptides which differ<br />
only by the substitution of D-tyrosine for L-tyrosine can be<br />
resolved. This example illustrates the effect of conformational<br />
differences on mobility, since CZE cannot separate<br />
isomers without chiral additives.<br />
48<br />
44<br />
40<br />
6 7<br />
Time [min]<br />
Figure 30<br />
CZE separation of neurotensin isomers<br />
Neurotensin amino sequence: pGlu-Leu-Try-Glu-<br />
Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-and pGlu-<br />
Leu-Try-Glu-Asn-Lys-Pro-Arg-Arg-Pro-D-Tyr-Ile-<br />
Leu.<br />
Conditions: 30 mM tetraborate, pH 8.3,<br />
V = 18 kV, i = 42 mA, l = 50 cm,<br />
L = 65 cm, id = 50 mm with 3X<br />
extended pathlength detection<br />
cell, l = 200 nm, peptide concentration<br />
2.5 × 10 -5 M each<br />
Determination of drugs and drug metabolites has also<br />
been successful by CZE. Figure 31a shows the analysis of<br />
Cefixime (CX), an oral cephalosporin antibiotic, and its<br />
metabolites. The strong hydrophilicity of these metabolites<br />
makes extraction from biological fluids difficult. For the<br />
same reason, separation by reversed phase liquid chromatography<br />
is insufficient, even with ion-pairing agents. Identification<br />
of the drug in human urine is shown in figure 31b.<br />
Quantitative analysis within about 3 % RSD was obtained.<br />
An example of the use of CZE for environmental analysis is<br />
shown in figure 32. In this study, conditions were optimized<br />
to separate aromatic sulfonic acids and related compounds.<br />
Here, 4-chlorobenzesulfonate is identified as a leachate at<br />
an environmental clean-up site in the U.S. Peak identification<br />
was confirmed by mass spectrometry. Along with<br />
60
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