Chemistry Reference
In-Depth Information
respect? The typical eluent in CE is a non-toxic salt solution, and the amount of this buffer is determined not
by the inner diameter of the capillary but by the total volume of the microvials that a human operator can
manipulate. This is commonly 100
l for typical commercial or research instruments, although smaller
volumes can be easily employed as well. For purposes of comparison, Table 9.1 (adapted from Noga
et al.
[14]). contains the inner diameters of columns, flow rates and volumes of eluent consumed during a 20 min
run for various modes of LC.
Table 9.1 reveals that only capillary HPLC (also known as nano-LC) is comparable with CE for eluent
economy. The other modes of HPLC consume much more eluent per run than does CE. This highlights a
problem associated with this technique. It is not easy to generate the flow rates necessary for capillary LC
with a typical HPLC pump, which can deliver no less than 100
μ
l min
-1
. The solution is to split the eluent just
after the pump. The splitter can be a simple 'T' connection or an active device that compensates for viscosity
changes during gradient elution. The major part of the flow is expelled as waste, and a small percentage is
introduced into a capillary column. Therefore, more than 99
μ
of an ultrapure, expensive eluent is wasted, so
nano-LC cannot be called a green method when splitters are used. A few manufacturers have recently
introduced splitless nano-LC instruments, which eliminate almost all the disadvantages of splitters. Such
solutions have been provided by Agilent Technologies (Santa Clara, California) and the Waters Corporation
(Milford, Massachusetts) [15]. Eksigent Technologies, Inc. (Livermore, California) [16] recently introduced
its commercial cHPipLC Nanoflex system. The glass chip contains etched, microfabricated channels with
C18 reversed-phase packing, which accommodates a direct coupling with a nano-HPLC pump and delivers
the mobile phase to the chip.
The eluent flow in a splitless nano-LC instrument is controlled as follows: flow meters in each mobile
phase path continuously monitor the flow rate and feed a signal back to a microprocessor. Pressure is
generated by connecting laboratory air or nitrogen to a pneumatic amplifier that increases the pressure in the
system. A microfluidic controller regulates this pressure to produce the required flow rate. For example, 100
psi of incoming air pressure from the laboratory air system can be used to generate a range of hydraulic
pressure up to 10 000 psi [16]. However, apart from the cost of nano-LC, the technology involved in nanoflow
pumping for HPLC mobile phases would sacrifice the portability and simplicity required of a truly green
analytical system.
Other innovations are being sought, in addition to using pressure to actuate the liquids. Much research and
development has been driven by the need to effectively manipulate small volumes of chemical and biological
liquids at micro and/or nano rates of flow. The principles of these pumping techniques are based on several well-
known phenomena. Electrokinetically-driven continuous flow pumps (such as electrophoretic and electroosmotic
pumps), surface chemistry-based continuous flow micropumps (such as the opto-electrowetting-based pump),
%
Table 9.1
Typical internal diameters, flow rates and amounts of eluent consumed for various LC columns.
Column ID
Common name
Typical flow rate
Amount of eluent consumed
(per 20 min run)
75
μ
m
Capillary
100-300 nl min
-1
2-6
μ
l
150, 250
μ
m
Capillary
300-500 nl min
-1
6-10
μ
l
500
μ
m
Capillary/narrowbore
0.5-10
μ
l min
-1
10-200
μ
l
0.75, 0.8 mm
Narrowbore/
microbore
10-100
μ
l min
-1
0.2-2 ml
1.1, 2.1 mm
Microanalytical
100-500
μ
l min
-1
2-10 ml
4.6 mm
Analytical
1-2.5 ml min
-1
20-50 ml
8 - 20 mm
Semi-preparative
>2 ml min
-1
>40 mls
