Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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Figure 1. Effect of gradient time on peak width (A) and peak capacity (B) using a 250 mm × 0.2 mm (closed symbols) and a 50 mm × 0.2 mm (open symbols) monolithic column. Sample: six-protein digest (transferrin, bovine serum albumin, �-galactosidase, alcohol dehydrogenase, lysozyme, and cytochrome c), 1 µL injection (0.5 pmol/ µL); flow rate: 2 µL/min; aqueous acetonitrile gradient from 1% to 35% with 0.05% TFA ion-pairing agent; column temperature: 60 °C. Detection at 214 nm using a3nLflowcell. of tG (Figure 1B). For gradient times below 60 min, the peak capacity of the 50 mm long monolith was higher than that of the 250 mm monolith. This can be explained by the difference in morphology of the two monoliths. 14 At undersampling conditions, the total peak capacity ( 2D nc) and total analysis time (ttot) in off-line two-dimensional LC are given by 1 tG 2D nc ) st t tot ) 1 t + × 1 tG s t 2 tG 2 W (1) · 2 t (2) where 2 tG is the second-dimension gradient time, 2 W is the average second-dimension peak width (equivalent to 4σ), 1 t is the first-dimension analysis time, 2 t is the second-dimension analysis time, and st is the sampling time. The first-dimensional analysis time is the sum of the column holdup time ( 1 t0), the dwell time ( 1 tdwell), and the gradient time ( 1 tG). The seconddimension analysis time is the sum of 2 tdesalt, 2 t0, 2 tdwell, a wash step ( 2 twash), and the second-dimension column equilibration time ( 2 teq), which corresponds to the time needed to flush the column with three column volumes to obtain good retention time stability. The peak production rate (�, min -1 ) is defined as 7018 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 � ) 2D nc Figure 2 illustrates the effect of the 2 tG on the � using a 50 mm and 250 mm long monolithic column. The 1 D RP separation was performed using a 50 mm × 1 mm monolithic column, applying a 10 min gradient at pH ) 8. The 2 D analysis included a preconcentration and desalting step on a monolithic trap column ( 2 tdesalt) and a RP gradient separation at pH ) 2onthe capillary column. Initially, a steep increase in � can be observed. In this region, � is dominated by the contributions of 2 t0 and 2 teq time to the total analysis time in the second dimension. With increasing gradient time, the peak capacity initially strongly increases (Figure 1B) and the peak-production rate reaches a maximum. When applying even longer gradients, the peak-production rate decreases. This is because the peak capacity marginally increases with the gradient time at longer gradient duration (see Figure 1B). The existence of a maximum can also be demonstrated by taking the derivative in eq 3 with respect to 2 tG. Forcing this derivative to be 0 yields 2 tG,max ) � a t tot b� s t( 1 td 1 tG + 1) + 2 t d where 2 tG,max refers to the gradient time in the second dimension that yields the highest value for �. 1 td ) 1 t0 + 1 tdwell, and 2 td ) 2 tdesalt + 2 t0 + 2 tdwell + 2 twash + 2 teq. The maximum peakproduction rate using the 50 mm long monolithic column is obtained at a 2 tG ∼ 9 min, and the maximum shifts to a higher Figure 2. Effect of 2 D gradient time on peak-production rate using 250 mm (a) and a 50 mm (b) long second-dimension monolithic column with a sampling time of 30 s (solid line), 50 mm column with sampling time of 60 s (- --; c), and 50 mm column with sampling time of 120 s (---; d). First-dimension separation on a 50 mm × 1 mm long column, 1 D delay time is 4 min, 10 min 1 D gradient time. 1 D gradient from 1% to 26% acetonitrile at pH ) 8 (10 mM aqueous ammonium carbonate buffer) at a flow rate of 60 µL/min. Seconddimension separation on a 250 mm column operating at a flow rate of 2 µL/min included a 1 min preconcentration, delay time of 0.4 min, t0 time of 3.53 min, 0.5 min wash step, and 10.6 min equilibration time; 2 D gradient from 1% to 35% with 0.05% TFA ion-pairing agent. Second-dimension separation on a 50 mm column operating at a flow rate of 2 µL/min included a 1 min preconcentration, delay time of 0.4 min, t0 time of 1.36 min, 0.5 min wash step, and 4.1 min equilibration time; 2 D gradient from 1% to 35% with 0.05% TFA ionpairing agent. (3) (4)
Figure 3. Peak-production rate versus total analysis time when either a 50 mm (closed symbols) or a 250 mm (open symbols) long 2 D monolithic column is used at optimal 2 tG and two or more fractions are collected. LC conditions described in Figure 2. value (20 min) when the 250 mm long monolith is used. This is because the a/b ratio increases with the column length (see Figure 1A); the slope of 2 tG vs 2 wb is lower for the 250 mm column, which implies that the b is lower. Also, the a decreases with column length. In addition, the � of the 50 mm long monolith is more than a factor of 2 higher than that of the 250 mm monolith. This is due to two effects. First, the region of 2 tG considered (from 1 to 60 min) contemplates situations in which the 250 mm column yields broader peaks than the 50 mm column (see Figure 1A), reducing 2 nc for the 250 mm column compared to the 50 mm monolith for a short gradient duration (Figure 1B). Second, with increasing column length, t0 and teq increase, affecting 2 td and, hence, the � (see eq 3). Figure 2 also shows that the optimal 2 tG depends on the sampling rate. This is clearly perceived from eq 4, where 2 tG,max depends on st. The higher st, the higher the value of 2 tG,max. Effect of Sampling Time on Peak-Production Rate. Figure 3 illustrates the � and total analysis time when either a 50 or a 250 mm long 2 D monolithic column is used at optimal 2 tG and two or more fractions are collected. When a 50 mm 2 D column is used, first, a strong increase in � is observed. At higher sampling rates, the increase levels off and � tends to reach a maximum around 10 peaks/min. When a 250 mm 2 D column is used, � increases slightly from 4.5 peaks/min (2 fractions) to a maximum of 5 peaks/min after sampling only three fractions. Apparently, when a 50 mm long 2 D column is used and a slow sampling rate is applied, � is significantly affected by the contribution of the 1 D analysis time to the total analysis time. With decreasing sampling time or when longer 2 D columns (longer t0 and teq) are used, � is dominated by the 2 D analysis time. Figure 4 shows the RP(pH)8)/×/RP(pH)2) separation of a sixprotein digest when applying a sampling time of 60 and 30 s, respectively. The 1 D separation was performed using a 50 mm × 1 mm monolithic column applying a gradient time of 10 min. The 2 D separation was executed on a 50 mm × 0.2 mm long monolithic column, applying a 2 tG of 7.5 min. The maximum theoretical peak capacity of the separation shown in Figure 4A is 1200, and the separation was completed in 98 min. The Figure 4. RP (pH ) 8)/×/RP (pH ) 2) separation of a digest of six proteins showing the effect of sampling time (st ) 60 s (A); st ) 30 s (B)) on peak capacity and total analysis time. 50 mm × 1mm 1 D column and 50 mm × 0.2 mm 2 D column. Further conditions as described in Figure 2. Figure 5. Off-line 2 D-LC separation of an E. coli digest using a sampling time of 15 s. 50 mm × 1mm 1 D column and 50 mm × 0.2 mm 2 D column. Further conditions as described in Figure 2. separation in Figure 4B yielded a maximum 2 D-LC peak capacity of 2400 in 164 min. Whereas the theoretical peak capacity doubles, the total analysis time increased only by 60%. It should be noted that the separation space was not completely filled. This is because the retention mechanisms of the reversedphase separations performed at pH ) 8 and 2, respectively, are not completely independent. As a consequence, the fraction of the total peak capacity that is actually used (“sample peak capacity”) is lower than the theoretical peak capacity. For the analysis of more complex samples, such as an E. coli digest, the most productive way to obtain a higher peak capacity in LC/×/LC, while still working at undersampling conditions, is to increase the number of 1 D fractions collected; see Figure 2. Increasing 2 tG is less effective. Figure 5 shows the LC/×/LC separation of E. coli digest. The sampling rate (15 s) was selected such that a maximum theoretical peak capacity of 6700 could be achieved within a total analysis time of 740 min. Trade-Off between 1 D-LC and the Optimized LC/×/LC Approach. Figure 6 illustrates the trade-off between the 1 D-LC system using a 50 mm and 250 mm long monolithic column and the RP(pH)8)/×/RP(pH)2) using a 50 mm long monolithic column in each dimension. The total 1 D-LC analysis time is the sum of the desalting time (0.5 min), the column holdup Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7019
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Anal. Chem. 2010, 82, 6745-6750 Let
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purchased from Invitrogen-Molecular
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Figure 3. Electropherograms of TPP-
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Anal. Chem. 2010, 82, 6751-6755 Res
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(pH 8.0) with cysteine and cystamin
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Figure 4. Ion mobility mass spectra
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GOx glucose + O298 gluconic acid +
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coater at 2000 rpm for 20 s, and th
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Figure 5. Comparison of cyclic volt
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in channels with either no grooves
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indicators of atmospheric processin
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Figure 4. SERS analysis of NAADP co
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Anal. Chem. 2010, 82, 6775-6781 Hig
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tion, 2 µL of proprionaldehyde wer
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Figure 4. Analysis of 2a by HPLC-MS
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eaction of 1a with PBH can be condu
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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Figure 4. Schematic representation
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esult in a big SPR signal change wi
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 1. Schematic view of the pre
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from the specimen and enclosed in a
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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of which was constant for all ampli
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Page 227 and 228: Figure 3. Equilibrium response as a
Page 229 and 230: Figure 6. The average measured resp
Page 231 and 232: for the fill time, and we find that
Page 233 and 234: nucleotide tails. 3-5 Thus, the amo
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Page 239 and 240: Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242: Figure 2. Configuration editor wind
Page 243 and 244: Figure 4. Dependencies between volu
Page 245 and 246: Since the time for liquid expulsion
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Page 249 and 250: Figure 1. Representation of the Car
Page 251 and 252: Table 2. Capillary Electrophoresis
Page 253 and 254: Figure 4. (A) Typical raw electroph
Page 255 and 256: field. DNA profiles were delivered
Page 257 and 258: Another approach to handle this lim
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Page 261 and 262: Figure 5. Relevant score plots and
Page 263 and 264: CONCLUSIONS In this paper we have i
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Page 267 and 268: Figure 2. Qdot-based FLFTS response
Page 269 and 270: Figure 6. Fluorescence imaging of Q
Page 271 and 272: Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273: Table 1. Effect of 1 D-LC Condition
Page 277 and 278: Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280: luciferase (T7 control vector) was
Page 281 and 282: Figure 3. Inhibitory effects of luc
Page 283 and 284: Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286: Electrochemistry. Electrochemical m
Page 287 and 288: Figure 2. SEM images of Au substrat
Page 289 and 290: Table 3. Film Thickness Measurement
Page 291 and 292: Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294: Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296: Figure 4. (A) Variation in the FL i
Page 297 and 298: Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300: Scheme 1. Proposed Mechanism for Fl
Page 301 and 302: one or more drawbacks including poo
Page 303 and 304: Figure 2. Free fluorophores do not
Page 305 and 306: Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308: Figure 2. Comparative analysis of d
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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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