Flow Imaging Microscopy: Characterizing Column Packing Materials

Chromatography is an essential technique for the separation and purification of complex mixtures. In this process, a sample that has been dissolved in a mobile phase is forced through an immiscible, immobile stationary phase. The phases are selected to ensure that sample components have varying solubilities in each phase.

A component that lacks solubility in the stationary phase but is highly soluble in the mobile phase will travel faster through it than a component that is quite soluble in the stationary phase.

These variations in mobility result in the separation of sample components as they move through the stationary phase. Chromatography frequently employs columns where the stationary phase is packed.

The shape and size of the particles in the packing material largely influence the mobility of various components within a sample. While popular particle analysis methods like laser diffraction, dynamic light scattering, sieving, and Coulter Counters will demonstrate the particle size distribution, they do not provide information on particle shape.

Manual microscopy has been utilized in the past to examine particle shape more closely. Microscopy is highly time consuming, and does not enable the analysis of statistically significant sample amounts.

Using Flow Imaging Microscopy for digital image analysis delivers crucial shape and size insight in column packing materials. This enables better management of column density, and in turn, improved control of column performance. It additionally allows damaged (non-spherical) particles to be traced, which are frequently found in various lots of packing material.


The FlowCam®, a flow imaging microscope, is optimally designed for the efficient characterization of both the particle shape and size of column packing materials. The FlowCam records and keeps a digital image of every particle in the sample and more than 40 unique measurements are performed for each image of a particle.

The instrument can collect, quantify, and store this particle information at a speed of up to 50,000 particles/ minute, generating much more statistically significant data compared to manual techniques in a significantly faster time.

Figure 1 presents the results of a standard FlowCam analysis of a polymer resin sample. As can be observed from the volume and frequency distributions (upper two graphs), the sample has a mean Equivalent Spherical Diameter (ESD, volume based) of 60.80 µm and a typical Gaussian size distribution.

FlowCam results for polymer resin sample.

Figure 1. FlowCam results for polymer resin sample. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.

In the lower left, the aspect ratio (width/length) scattergram shows that the particles are not homogeneous in shape. Particles of varying shapes and sizes can be visualized with ease using the FlowCam's VisualSpreadsheet® software.

Screen shots depicting two sets of particles are shown in Figures 2 and 3, which are determined to be acceptable and unacceptable. A round shape is seen in the first set of particles shown in Figure 2.

The ruler in the lower right-hand corner shows that each of these particles has an ESD right around the 60.92 µm mean for the sample. Figure 3 shows the second set of particles that are not as round and seem to be contamination, misshapen beads, or pieces of damaged beads.

Round “acceptable” particle images.

Figure 2. Round “acceptable” particle images. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.

Less round “unacceptable” particle images.

Figure 3. Less round “unacceptable” particle images. Image Credit: Yokogawa Fluid Imaging Technologies, Inc.

For the intended application in this specific batch of polymer resin, the aim was that more than 95% (by volume) of the batch should consist of the type of particles depicted in the first example (within 15% of the size mean and very round).

As the allowable percentage of the second particle type (erose shape) is so small (5%), a highly time-consuming manual microscope technique would be required to make a statistically significant evaluation of whether the batch meets the requirements.

Aspect ratio is one of the 40+ measurements collected by FlowCam for every particle. Aspect ratio is a measurement of the shape of the particle (from a 2D projected image) and is calculated as length/width.

A user who is familiar with the sample and its appearance under a microscope can efficiently evaluate the particle images using the interactive capabilities of VisualSpreadsheet to determine the aspect ratio value at which the particles should no longer be classified as acceptable.

It was decided for this batch that only particles having an aspect ratio of more than .85 and being within 15% of the mean in size would be classed as acceptable. Software filters can then be easily produced for the unacceptable and acceptable particles.

The filters are added to the main statistics screen once they have been defined in VisualSpreadsheet. Based on these filters, all particles are automatically categorized as either unacceptable or acceptable.

Each particle is segmented into the two categories in real time during acquisition by the FlowCam, producing the desired statistics (% of each particle type by volume) instantly after the sample run is complete. The filters can be stored and reused on new samples at any point in the future.

Results and Conclusions

Figure 4 presents the sample run results as before, but this time displays the filter statistics for unacceptable and acceptable particles. The summary statistics demonstrate that for a total of 9,261 particles imaged, saved, and quantified, 340 of them (2.74% by volume) were categorized as unacceptable and 8,921 of them (97.26% by volume) were categorized as acceptable.

As more than 95% of the particles have an acceptable shape, this means that this batch is considered to be acceptable. At this stage, the summary statistics can be exported to any LIMS system or database as required. All particles can be viewed according to category if the operator clicks on the filtered results and instructs the software to show those images alone.

Automated filter results (bottom).

Figure 4. Automated filter results (bottom). Image Credit: Yokogawa Fluid Imaging Technologies, Inc.

In summary, VisualSpreadsheet and the FlowCam were able to measure 9,261 particles automatically in just over a minute in order to determine whether this polymer resin batch adhered to the required specifications.

Employing manual inspection via a microscope would have taken hours to make the same characterization. Along with this, the manual technique would not have generated results with equivalent precision or statistical significance.

This information has been sourced, reviewed and adapted from materials provided by Yokogawa Fluid Imaging Technologies, Inc.

For more information on this source, please visit Yokogawa Fluid Imaging Technologies, Inc.


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