7. Wada invented the first Couette flow cell in 1964 [23, 24]. The
sample is “endlessly” and stably flowed between two quartz
cylinders one of which rotates and one of which is stationary,
which enables extended data collection. Couette cells designed
to use quartz capillaries and rods and an annular gap of 250 μm
reduce the sample volume to less than tens of microliters
(Fig. 4) [8].
8. Peptide/liposome samples have broad bands, so increasing the
light throughput by choosing 2 nm bandwidth seldom changes
the spectral shape. It is usually necessary to average over a
number of spectra. As a rule of thumb, the true instrument
high tension voltage of the spectropolarimeter should usually
be kept below 600 V—otherwise too many of the incident
photons are being absorbed or scattered and the LD reading
will be nonsense. It should be noted that stray light is often
introduced in flow LD experiments when the power cable is
inserted. The photomultiplier tube will happily count these
photons but it is not indicative of satisfactory instrument performance because what we need to measure is light passing
from the monochromator to the detector. Make sure you check
for any instrument error flags.
9. If the stationary baseline of water is always the same (relative to
the signal magnitude) when the capillary is reinserted in a
different position, then one can assume the baseline is independent of orientation of the capillary.
10. In an ideal world, all three baselines will be identical. However,
water, stationary liposomes and liposomes in shear flow seldom
have the same scattering. The liposome options usually cancel
the scattering contribution to the signal better than the water,
though it should be noted that liposomes in stationary cells or
without the analyte may be different in size or shape from the
rotating sample. Often the only way to get a good baseline
correction is to repeat the experiment multiple times and use
the baseline overlay outside the absorbance region to choose
the best pair of sample and baseline spectra.
11. Although the absorbance of the lipids themselves can usually
be ignored, lipids often have light absorbing impurities. In
addition, the scattering increases with decreasing wavelength.
12. If literature data are not available, the absorbance spectrum of
the chromophores may need to be collected independently.
Film LD or a calculation may be needed to provide transition
polarization assignments. As illustrated in Fig. 8, FMOC has
significant approximately long-axis polarized intensity at
260 nm and its 210 nm transition is closer to short-axis polarized. For film orientation we may write:
Flow LD of Protein–Membrane Systems
461
sample is “endlessly” and stably flowed between two quartz
cylinders one of which rotates and one of which is stationary,
which enables extended data collection. Couette cells designed
to use quartz capillaries and rods and an annular gap of 250 μm
reduce the sample volume to less than tens of microliters
(Fig. 4) [8].
8. Peptide/liposome samples have broad bands, so increasing the
light throughput by choosing 2 nm bandwidth seldom changes
the spectral shape. It is usually necessary to average over a
number of spectra. As a rule of thumb, the true instrument
high tension voltage of the spectropolarimeter should usually
be kept below 600 V—otherwise too many of the incident
photons are being absorbed or scattered and the LD reading
will be nonsense. It should be noted that stray light is often
introduced in flow LD experiments when the power cable is
inserted. The photomultiplier tube will happily count these
photons but it is not indicative of satisfactory instrument performance because what we need to measure is light passing
from the monochromator to the detector. Make sure you check
for any instrument error flags.
9. If the stationary baseline of water is always the same (relative to
the signal magnitude) when the capillary is reinserted in a
different position, then one can assume the baseline is independent of orientation of the capillary.
10. In an ideal world, all three baselines will be identical. However,
water, stationary liposomes and liposomes in shear flow seldom
have the same scattering. The liposome options usually cancel
the scattering contribution to the signal better than the water,
though it should be noted that liposomes in stationary cells or
without the analyte may be different in size or shape from the
rotating sample. Often the only way to get a good baseline
correction is to repeat the experiment multiple times and use
the baseline overlay outside the absorbance region to choose
the best pair of sample and baseline spectra.
11. Although the absorbance of the lipids themselves can usually
be ignored, lipids often have light absorbing impurities. In
addition, the scattering increases with decreasing wavelength.
12. If literature data are not available, the absorbance spectrum of
the chromophores may need to be collected independently.
Film LD or a calculation may be needed to provide transition
polarization assignments. As illustrated in Fig. 8, FMOC has
significant approximately long-axis polarized intensity at
260 nm and its 210 nm transition is closer to short-axis polarized. For film orientation we may write:
Flow LD of Protein–Membrane Systems
461