A separation method utilized in numerous areas of analytical chemistry to separate, identify, and qualify various compounds in a specific solution is named High Performance Liquid Chromatography (or High Pressure Liquid Chromatography).
HPLC uses a column that holds chromatographic packing material that retains molecules as they are pumped through the column. The retention time is dependent on the carrier liquid (solvent), packing material, and the particular type of molecule. Each chemical species in the injected sample is most commonly identified by UV and Mass Spec detection.
Most traditional analytical HPLC packing materials are usually alumina or silica-based material, and have an average diameter of between 2 and 30 microns. The particle size of the packing material is typically calculated by employing techniques that assume all particles are uniformly spherical.
This is not always the case, for example, in the manufacture of the silica and bonding, fines and irregular-shaped particles can make up part of the final product. As particle size is reduced, the impact of fines and irregular-shaped particles warrant a higher degree of control on the incoming quality of the silica material.
Variation in particle shape can directly influence reproducible performance. Filling stage back pressure of the column can differ greatly depending on the shape of the HPLC particles. The flow of a packed HPLC sample column utilizing perfectly spherical particles has an extremely predictable performance when employed to separate molecules.
The manufacturing of a column with truly spherical particles would be predictably consistent. Yet, in many cases, some of the particles used for HPLC column packing exist as fines or are irregular in shape. The packing geometry can become irregular as a result, and it may express as non-uniform performance of the final product from column to column and lot to lot.
A column packed with uniform spherical beads is easier to manufacture and yields consistent and uniform results. The same column containing irregular particles and fines will present manufacturing challenges as well as end-user concerns with respect to consistency.
The amount of irregular packing and fines can change the performance dependability of the column and drive HPLC columns to become a lot more costly to produce due to the extra expertise and care required to achieve consistency in production. Any potential downtime in production can lead to lots of materials containing different percentages of irregular particles.
Theoretically, if a technique for the packing material manufacturer to specify shape characteristics and for column manufacturers to inspect incoming materials against those specifications existed, creation of HPLC columns would be more predictable, cheaper and give consistent performance.
The answer could be in recent advancements in image analysis. The analytical method employed in the experiment outlined in this article is Dynamic Image Analysis. Using image analysis, as particles pass through a detection zone, images are gathered and analyzed. For all dynamic image analysis instruments available on the market today, ISO 13322-2 is used as the guideline.
The main advantage of employing image analysis for HPLC column packing materials is to allow the identification and quantification of the different particle shapes present in incoming materials.
Throughout the manufacture of specific HPLC columns, inconsistent packing pressures seen, and a wide variation in their performance instigated the analysis of this particular sample. For this experiment, the SentinelPro Particle Shape Analyzer measured incoming HPLC materials utilized to pack Size Exclusion Chromatography columns.
Correct sampling of the incoming materials performed to verify that a homogeneous sub-sample was collected. The sample was then suspended in water and analyzed. Much like laser diffraction systems, the SentinelPro system recirculates the sample. All of the particles are measured and oriented randomly in order to measure all dimensions, not just two sides.
Random thumbnail images of the alumina sample from the 10,000 captured in just over one minute. The value under each thumbnail represents the Feret Aspect Ratio of each particle.
Results and Discussion
It was apparent that this alumina sample possessed two distinct shape populations after the analysis and review of all particle thumbnail images. One was rod-shaped and the second had an aspect ratio near unity, but the particles were not round. These rod-like and irregular particles could not be differentiated if the sample had been analyzed by utilizing a sizing method which assumes all particles to be spherical.
In developing a technique for this sample, particles with an aspect ratio over 1.9 were considered to be rod-like and seen as more likely to negatively affect the packing efficiency of the column. This aspect ratio value threshold was acquired from sample lots that did and did not display the problems described previously.
The percentage of rod-like particles in this column material was 15.90% of the total distribution as seen in the histogram and statistical data, which, compared to the historical lot of good material, was shown to be too much for the particular application of the column.
Comprehensive Statistics for the alumina sample.
A review of the thumbnails for the particles that possess a Feret Aspect Ratio of above 1.9 provides a better perspective on what these particles look like. With a technique established to ascertain that the Feret Aspect Ratio of incoming alumina packing material had less than 10% rod-like particles, the end-user could create a simple process control indicator with the Particle Insight.
A sampling of the thumbnails of the 1,591 particles found to have an aspect ratio greater than 1.9.
The Percentile Statistics feature enables you to see particle statistics and pass/fail criteria.
By utilizing the Percentile Statistics feature, incoming quality control can easily and quickly determine if a lot of material passes or fails the criteria decided by management.
With its Dynamic Image Analysis technique, the Particle Insight can help to decrease costs by identifying HPLC packing material inconsistencies before reaching column manufacturing. The manufacturer is still responsible for establishing the acceptable percentage of non-spherical particles for incoming materials.
Once the manufacturer determines the incoming quality control criteria, Particle Insight can be employed as a pass/fail inspection tool to establish the percentage of non-spherical to spherical particles. Solving this potential problem at the start of the process enhances production and helps to ensure consistent HPLC column performance and quality.
This information has been sourced, reviewed and adapted from materials provided by Particulate Systems.
For more information on this source, please visit Particulate Systems.