Particle Size Reference Standards
FFF theory is well-developed for the separation and sizing of polydisperse particles in simple matrices using retention times
(10). Interpreting peak areas to determine concentrations is somewhat more difficult. By using conventional detection, such
as UV absorbance, refractive index, light scattering or fluorescence, the technique can use stable dispersion calibration
reference standards such as NIST-traceable polystyrene beads of known particle sizes. Unfortunately, the detection of polystyrene
(carbon) beads is not practical by ICP-MS. For that reason, when external size calibration is required, an in-line conventional,
nondestructive detector before the ICP-MS detector is a relatively simple addition to the instrumental setup, as shown in
Figure 3. As new reference materials become more available, polystyrene bead standards can be replaced with metallic nanoparticles.
Currently, NIST provides monodisperse (same size and shape) gold and silver nanoparticles suitable for sizing at trace levels.
The benefit of using ICP-MS as a detection system for FFF compared to UV absorbance can be seen in Figure 4, which shows the
separation of mixed gold and silver nanoparticles. It can be seen that when using UV detection alone (inset graph), the fractogram
does not differentiate between the silver and gold particles and only shows the FFF separated particles by size (10, 30 and
60 nm). The main fractogram obtained using ICP-MS detection clearly shows the separated particles and the signal intensities
for both the silver and gold particles at their atomic masses of 107 amu and 197 amu, respectively (7).
Figure 4: FFF fractogram of the separation of mixed silver and gold nanoparticles, using both UV absorbance (inset graph)
and ICP-MS detection. Adapted from reference 7.
External size calibration also requires stable particle dispersions and minimal membrane interactions. For that reason, a
surfactant may be needed. In some cases the surfactant most suitable for metal-containing nanoparticles may not be compatible
with polystyrene standards, again suggesting that other size calibration standards would be useful. Additionally, when using
ICP-MS on-line detection, other factors need to be considered with respect to choice of carrier solution. For example, the
carrier solution can cause salt formation on the ICP-MS cones, leading to instability and a decrease in signal intensity over
time. Another potential problem is that polyatomic spectral interferences can be created in the plasma by components in the
carrier solution. For example, the use of chloride-based salts can generate interferences for elements such as vanadium, chromium,
arsenic and selenium, among others. So even though modern ICP-MS instrumentation often includes a collision–reaction cell
to minimize these interferences (11), the optimization of FFF separation conditions must also consider the impact on the ICP-MS
In addition, the correct implementation of both internal and external calibration standards is critical in ICP-MS. Typically,
internal standards are prepared in 1–5% acid and added to all calibration standards and samples. Unfortunately, the introduction
of a dissolved metal standard into a near-neutral-pH FFF mobile phase can result in precipitation of analytes resulting in
inaccurate metal quantification of fractionated samples. An alternative approach is to use split flows that allow the introduction
of acidified internal standards directly to the ICP nebulizer after elution from the FFF channel. Internal standards have
also been mixed directly into the carrier fluid and simultaneously used for external calibration by comparison of elemental
response factors to the internal standard (12).
Alternative calibration approaches for FFF–ICP-MS studies include injecting a known mass of metal using a flow-injection technique
and comparing the area of the known mass to the area of the unknown sample elution peak. External calibration can also be
performed by analysing a continuous flow (delivered by a flow ratematched peristaltic pump) of known concentrations of metal,
developing a calibration curve and then converting the intensity of the fractogram reading to a concentration value (13).