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250
are stored in the computer memory and processed separately. Since a
MALLS detector detects the scattered light simultaneously at up to 18 angles
(ca. 30-150
°
), each slice gives rise to (up to) 20 data points:
V
i
The elution volume (ml)
c
i
:
The concentration (from the concentration sensitive detector)
R
i,1
-R
i,18
The Rayleigh factors for each of the 18 angles.
The data matrix thus contains
i
x 20 data points (
i
= 1000-5000).
The software the performs the following calculations for each elution slice (
i
):
a)
First, the concentration is calculated from the baseline-subtracted
voltage (U
i
–U
0
) of the RI detector. Interestingly, dn/dc enters this
calculation in addition to an instrument constant (dn/dV). The latter is
obtained by separate calibration of the RI detector with a reference
substance with known refractive index, usually NaCl standards.
c
i
=
U
i
−
U
0
(
)
n
−
n
0
U
i
−
U
0
⎛
⎝⎜
⎞
⎠⎟
c
i
−
c
0
n
i
−
n
0
⎛
⎝⎜
⎞
⎠⎟ =
U
i
−
U
0
(
)
dn
dU
⎛
⎝⎜
⎞
⎠⎟
dn
dc
⎛
⎝⎜
⎞
⎠⎟
−
1
b)
Further, Kc
i
/R
θ
is calculated for each angle (
θ
) and a plot of Kc
i
/R
θ
versus sin
2
(
θ
/2) is then constructed (for each slice). Note this is
identical to Step. 1 in the conventional light scattering processing. An
example (‘Debye plot’ from Astra software) is shown below.