Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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
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