HPLC methods particle size distribution

Silica stationary phases display some ion exchange properties, which may also influence the separation characteristics of silica. One of the main disadvantages of the use of silica and silica-based stationary phases is their instability even at slightly alkaline pH, such as 8.0. HPLC stationary phases can be characterized with the average particle diameter and the distribution of particle size. Smaller average diameter and narrow particle size distribution generally enhances the efficacy of separation. The average particle diameter can be calculated with different methods ... 

The particle size distribution of packing materials has been shown to vary not only from one manufacturer to another but also with the measurement method used. For HPLC specifications, it is normal for manufacturers to give a particle size distribution based on an area distribution. However, this distribution has been pointed out [30] not to give a true distribution of the fine material... 

Before polyacrylamides are sold, the amount of residual acrylamide is determined. In one method, the monomer is extracted from the polymer and the acrylamide content is determined by7 hplc (153). A second method is based on analysis by7 cationic exchange chromatography (154). For dry7 products the particle size distribution can be quickly determined by7 use of a shaker and a series of test sieves. Batches with small particles can present a dust hazard. The percentage of insoluble material is determined in both dry and emulsion products. 

A strong positive feature of SEC is that instrumentation is readily available in the form of HPLC apparatus. No special experience is needed for those acquainted with this widely practiced method. Relatively unskilled operators can quickly learn to perform the analysis satisfactorily. Average particle sizes are quickly measured by the peak-position method. However, it is also feasible to determine particle-size distributions if appropriate computer software is available. Separation times are predetermined, because all species elute between the total exclusion and total permeation volumes (provided the desired SEC process is the only retention). No special method development is required, other than ensuring that the proper mobile phase-stationary phase combination is selected. Particle diameter is directly a function of retention or elution times. 

To interpret the results from pore size distribution measurements, it is usually assumed that the pores are tubular. However, this is far from the truth. The nature of the pores depends to some degree on the method used to synthesize the particles, but in all cases they form an interconnected network of irregularshaped channels. For most preparations the nature of the pore network is much like the interstitial space formed in a packed bed. Electron microscopy is used to examine the internal structure of HPLC packings. Except for some special cases, the internal structure is homogeneous throughout the particle. 

The number of theoretical plates is proportional to the column length and inversely proportional to the particle size. The advantage of using small particles is that they distribute flow more uniformly and, as a result, reduce the eddy diffusion, term A in the Van Deemter equation. However, the smaller particles increase the diffusional resistance of the solvent as well as the pressure drop (for a given flow rate). Choosing the flow rate is a critical parameter in developing an HPLC method. Low flow rates allow the analyte sufficient time to interact with the stationary phase and will affect both the B and C terms of the Van Deemter equation. 

Another excellent demonstration of the wide application range of CEC has been the microanalytical determination of the molecular mass distribution of celluloses in the investigation of cellulose-based objects of cultural and historical value [168]. Stol et al. employed a column packed with bare silica particles of 5 pm diameter with a nominal pore size of 300 A to achieve the separation of 2-kDa to 500-kDa celluloses by size exclusion (SE) CEC. The higher speed and smaller sample size advantages of SE CEC compared to SE HPLC distinguished CEC as the method of choice for the study of cellulose-based objects of archeological importance. 

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