Nano flow HPLC (<0.1 mm ID, flow-rate < uL/min) is essentially not different
from classical HPLC (4.6 mm ID column ~1 mL flow rate). However, it is
now widely recognised that the sensitivity of detection in HPLC by
UV, ESI-MS and other concentration dependent detectors is improved by
reducing the interior diameter of the column. This holds only if equal
amounts of a sample is introduced into the column. The gain in
sensitivity follows directly from the smaller volume i.e
the higher concentration
in which analytes
elute from the column. The gain is
proportional to the square of the ID ratio of the two columns.
The table compares the relative sensitivity of different columns
of different ID. Going from the conventional column of 4.6 mm ID down to
a 0.01 (10 um), the gain can be as large as over a factor of
200,000 times. High sensitivities are particularly relevant in
cases where sample amount is the limiting factor. Draw backs of nano
flow LC are (1) Nanoscale columns allow a very limited sample volume
injection and (2) difficulties associated with performing high
performance separations at the nano flow scale. The problem of limited
sample volume injections can be solved by the use of a solid phase
extraction column. SPE columns allow relatively large injection volumes
( e.g 5-50 uL) in a reasonable time interval (1-10 min). Maintaining the
chromatographic integrity in nanoflow LC requires a.o the elimination of
dispersing volumes, both upstream and downstream the analytical column.
gain ~ (d1/d2)2
|
Column |
Flow |
Relative |
|
ID |
rate |
Sensitivity |
|
(mm)> |
(uL/min) |
(-) |
|
| 4.6 |
1000 |
1 |
| 2 |
200 |
5.3 |
| 1 |
50 |
21 |
| 0.5 |
12 |
84 |
| 0.2 |
2 |
529 |
| 0.1 |
0.500 |
2,116 |
| 0.075 |
0.250 |
3,762 |
| 0.050 |
0.120 |
8,464 |
| 0.025 |
0.030 |
33,856 |
| 0.010 |
0.005 |
211,600 |
Instrumental demands for
high performance nanoflow HPLC include:
(i)
the HPLC pump must be capable of delivering a linear gradient
at the flow-rate of the column (see pump
hold-up volume)
(ii) the solid phase
extraction trap should not affect the separation efficiency (see
precolumn void volumes)
(iii) peak broadening in the ESI
interface should be absent or minimal (see
post column void volumes).