Properties of polymer-based columns
The selection of a suitable HPLC column for a specific application is a crucial decision in order to obtain the best possible results regarding separation, resolution, costs, analysis time, etc.
During this selection is important to take into account the base material/stationary phase (it is like the “foundation of a building”) which in the case of HPLC columns can be silica, polymer, etc. Anyhow, we should also take into account the separation mode and therefore, the selection of the functional group and/or analyte without forgetting the application area.
While it is true that silica-based columns work with most applications, they have several limitations which are:
Vulnerable at extreme pH: most columns only provide results between pH 2 to 8.
Silanolic activity, which means analyte interaction with the remaining silanol groups and peak tailing as a result. Even the columns sold as “fully end-capped” are not 100% silanol-free, and therefore they cannot provide an accurate result, especially when basic compounds (e.g. amines) have to be analyzed.
These undesired aspects of silica columns can be only solved by exchanging the base material, i.e. by choosing a more pH stable and silanol-free material.
The most used base materials alternatively to silica are polymers. There are several types of polymers intended for the same aim, these are the following:
Styrene divinylbenzene copolymer: eminently non-polar.
Polymethacrylate: more polar than the previous one.
Polyvinyl alcohol and polyhydroxymethacrylate: they can be functionalized with C18, amino groups, etc.
Usually, the polymer-based HPLC columns show several advantages over the silica-based ones, especially in the cases where silica-based cannot be used because of their limitations:
The pH stability for polymer-based columns is mostly pH 2-13.
Polymer-based columns have a longer lifetime because the material itself shows strong chemical resistance. This means a reduced price per injection in the end.
Very low or negligible bleeding which makes them appropriate for MS, LS and CAD detection.
Selection by HPLC Separation Mode
Liquid chromatography (LC) uses liquid as mobile phase (eluent). It is an analytical method that separates a mixture of compounds based on their physical and chemical differences.
High performance liquid chromatography (HPLC) is a method that introduces the mobile phase under high-pressure conditions resulting in rapid and high-performance separations.
The various interactions between the analyte, stationary phase (packing material), and mobile phase are the key factors for the separation.
A wide variety of separation modes can be achieved by using particular combinations of stationary and mobile phases. The following table gives an overview about most common used separation techniques and a short explanation of those.
• Separation is based on the partition equilibrium between stationary phase and mobile phase.
• The polarity of the stationary phase is lower than that of the mobile phase.
• Typically the mobile phase contains a mixture of organic solvents (methanol, acetonitrile, or THF) and
aqueous solvents (water or buffer).
• Use of lower polarity mobile phases fasten the elution.
• Separation is based on hydrophilic interaction.
• A high polarity stationary phase is used.
• Typically the mobile phase contains a mixture of organic solvents such as acetonitrile and aqueous
solvents (water or buffer).
• Using the higher polarity mobile phase causes a faster elution.
• Applicable for the analysis of high polar substances.
Gel Permeation Chromatography
• Network or pores on the surface of the packing material works as molecular sieve to separate molecules
based on their sizes.
• To separate molecules solely based on their sizes, it requires an analytical condition without any
compounds and packing gel interaction.
• The bigger the molecule size, the faster the elution sequence.
• Used for molecular weight or molecular distribution determination of macromolecules and qualification of
• Separation is based on electrostatic interactions between the ion exchanger and ionic solutes.
• The mobile phase of choice should have a sufficient buffering capacity at the pH that produces the largest
charge differences between the analyte of interest.
• The elution position is optimized by varying the pH, salt concentration, and/or ionic strength of the mobile
General Precautions for Column Handling
For the best performance of the column, please follow the instructions given below.
HPLC System Preparation
• Wash entire LC system prior to the column installation, including all flow-lines and sample loop by switching the valve, and then replace the washing solution with the eluent to be used.
• If desired new eluent has low miscibility/solubility to the eluent of previous analysis, first use the eluent that is miscible/soluble to both eluents, and then replace it with the desired eluent.
*If the eluent left in the system is not compatible with the column to be used, it may damage the column.
*A drastic change in the eluent compositions may remove substances adsorbed on the system and they may enter and deteriorate the column.
• Connect the column to LC system by following the “flow direction arrow” ( ) indicated on the column name tag. If guard column is used, position the guard column in front (before the inlet) of the analytical column.
• Make sure to insert the tubing all the way to the end fitting and secure it with the male nut. It is important that there is no extra space between the tubing and the column side of the end fitting. Presence of an extra space will let the sample to spread out and may result in wide peaks.
• Set the initial flow rate at less than half of the recommended flow rate and start the system. If using the column at an elevated temperature, keep a low flow rate until the temperature of the column reaches to the set temperature, and then gradually increase the flow rate to the desired.
*Verify that there is no solvent leak. It may cause electronic leakage, rust, and/or chemical injury.
*Make sure not to let air bubbles enter the column while installing the column. The air bubbles may damage the column.
* When restarting the system after column installation or after holding the eluent flow, start the system at less than half of the recommended flow rate. A rapid increase in pressure can damage the column.
* If the column was used at an elevated temperature, lower the flow rate to less than half of the recommended flow rate at the end of analysis. Then, turn off the column oven, and let the column temperature return to room temperature before stopping the pump. If the pump was stopped while the eluent inside the column was still hot, as the eluent temperature decreases, its volume also decreases. This may result in creating an empty space in the column and deteriorates the column.
*It is recommended to set the pump limiter to avoid exceeding the maximum pressure.
• When replacing the solvent, start the system at less than half of the recommended flow rate. Recommended solvent volume to introduce at each step is 3 to 5 times of the column volume.
• Check miscibility/solubility of the desired new solvent and the solvent currently filled in the column.
• When replacing the current solvent with a solvent with low miscibility/solubility to the current solvent, first use a solvent that is miscible/soluble to both eluents, and then replace it with the new solvent.
• When using a gradient method, changes in the eluent compositions may increase the column backpressure. Adjust the flow rate and column temperature so that the column backpressure remains below the usable maximum pressure.
• Remove the column from the system after replacing the in-column solvent with the shipping solvent. Securely tighten the end caps and store the column at a location with stable temperature (a cool and dark space is recommended).
*Never allow inside of the column to dry. It can damage the column.
• Do not remove end fittings.
• Do not make a strong impact on the column. Do not drop or hit the column on a hard surface.
*Read the operation manual before using the column.
Inspection method is described in the Certificate of Analysis (CoA).
Theoretical Plate Number (N) and Asymmetry Factor (Fas) were calculated using the below equations.
• Theoretical Plate Number (N)
• Asymmetry Factor (Fas)