Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Figure 3. Peak-production rate versus total analysis time when either<br />
a 50 mm (closed symbols) or a 250 mm (open symbols) long 2 D<br />
monolithic column is used at optimal 2 tG and two or more fractions<br />
are collected. LC conditions described in Figure 2.<br />
value (20 min) when the 250 mm long monolith is used. This<br />
is because the a/b ratio increases with the column length (see<br />
Figure 1A); the slope of 2 tG vs 2 wb is lower for the 250 mm<br />
column, which implies that the b is lower. Also, the a decreases<br />
with column length.<br />
In addition, the � of the 50 mm long monolith is more than a<br />
factor of 2 higher than that of the 250 mm monolith. This is due<br />
to two effects. First, the region of 2 tG considered (from 1 to 60<br />
min) contemplates situations in which the 250 mm column<br />
yields broader peaks than the 50 mm column (see Figure 1A),<br />
reducing 2 nc for the 250 mm column compared to the 50 mm<br />
monolith for a short gradient duration (Figure 1B). Second, with<br />
increasing column length, t0 and teq increase, affecting 2 td and,<br />
hence, the � (see eq 3). Figure 2 also shows that the optimal 2 tG<br />
depends on the sampling rate. This is clearly perceived from<br />
eq 4, where 2 tG,max depends on st. The higher st, the higher the<br />
value of 2 tG,max.<br />
Effect of Sampling Time on Peak-Production Rate. Figure<br />
3 illustrates the � and total analysis time when either a 50 or a<br />
250 mm long 2 D monolithic column is used at optimal 2 tG and<br />
two or more fractions are collected.<br />
When a 50 mm 2 D column is used, first, a strong increase<br />
in � is observed. At higher sampling rates, the increase levels<br />
off and � tends to reach a maximum around 10 peaks/min.<br />
When a 250 mm 2 D column is used, � increases slightly from<br />
4.5 peaks/min (2 fractions) to a maximum of 5 peaks/min after<br />
sampling only three fractions. Apparently, when a 50 mm long<br />
2 D column is used and a slow sampling rate is applied, � is<br />
significantly affected by the contribution of the 1 D analysis time<br />
to the total analysis time. With decreasing sampling time or<br />
when longer 2 D columns (longer t0 and teq) are used, � is<br />
dominated by the 2 D analysis time.<br />
Figure 4 shows the RP(pH)8)/×/RP(pH)2) separation of a sixprotein<br />
digest when applying a sampling time of 60 and 30 s,<br />
respectively. The 1 D separation was performed using a 50 mm<br />
× 1 mm monolithic column applying a gradient time of 10 min.<br />
The 2 D separation was executed on a 50 mm × 0.2 mm long<br />
monolithic column, applying a 2 tG of 7.5 min. The maximum<br />
theoretical peak capacity of the separation shown in Figure 4A<br />
is 1200, and the separation was completed in 98 min. The<br />
Figure 4. RP (pH ) 8)/×/RP (pH ) 2) separation of a digest of six<br />
proteins showing the effect of sampling time (st ) 60 s (A); st ) 30 s<br />
(B)) on peak capacity and total analysis time. 50 mm × 1mm 1 D<br />
column and 50 mm × 0.2 mm 2 D column. Further conditions as<br />
described in Figure 2.<br />
Figure 5. Off-line 2 D-LC separation of an E. coli digest using a<br />
sampling time of 15 s. 50 mm × 1mm 1 D column and 50 mm × 0.2<br />
mm 2 D column. Further conditions as described in Figure 2.<br />
separation in Figure 4B yielded a maximum 2 D-LC peak capacity<br />
of 2400 in 164 min. Whereas the theoretical peak capacity<br />
doubles, the total analysis time increased only by 60%. It should<br />
be noted that the separation space was not completely filled.<br />
This is because the retention mechanisms of the reversedphase<br />
separations performed at pH ) 8 and 2, respectively,<br />
are not completely independent. As a consequence, the fraction<br />
of the total peak capacity that is actually used (“sample peak<br />
capacity”) is lower than the theoretical peak capacity.<br />
For the analysis of more complex samples, such as an E. coli<br />
digest, the most productive way to obtain a higher peak capacity<br />
in LC/×/LC, while still working at undersampling conditions, is<br />
to increase the number of 1 D fractions collected; see Figure 2.<br />
Increasing 2 tG is less effective. Figure 5 shows the LC/×/LC<br />
separation of E. coli digest. The sampling rate (15 s) was selected<br />
such that a maximum theoretical peak capacity of 6700 could be<br />
achieved within a total analysis time of 740 min.<br />
Trade-Off between 1 D-LC and the Optimized LC/×/LC<br />
Approach. Figure 6 illustrates the trade-off between the 1 D-LC<br />
system using a 50 mm and 250 mm long monolithic column<br />
and the RP(pH)8)/×/RP(pH)2) using a 50 mm long monolithic<br />
column in each dimension. The total 1 D-LC analysis time is<br />
the sum of the desalting time (0.5 min), the column holdup<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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