5 Misconceptions About HPLC Columns
HPLC column is one of the very important components, which can generally be divided into three categories: reversed-phase column, normal-phase column, and other columns.
① Reversed-phase column: it is the most used column; generally about 70%; among them, C18 HPLC column accounts for about 65%; C8 HPLC column accounts for about 15%; phenyl HPLC column <5%. C18 is the most widely used in reversed-phase chromatography. About more than half of them.
②Normal phase columns: Normal phase columns are also frequently used, and they account for about 20%.
③Other chromatographic columns (chiral columns, ion exchange columns): about 10%.
Today, HAWACH will discuss the five misconceptions about HPLC columns.
1) Higher efficiency is always achieved with columns filled with smaller particles
Although for well-filled columns the efficiency increases as the average diameter of the particles in the packed column decreases, the full performance of the column will not be achieved if the off-column contribution to the band broadening of a particular HPLC hardware system is high enough. The extra-column component includes all volumes other than the packing itself. These contributions include the following.
1. The inlet volume includes the loop size as well as the other volumes within the inlet valve.
2. The volume of the tubing from the inlet of the injector to the inlet of the column.
3. The volume of the protective column gap including end connections (if any).
4. In-line filter volume (if any).
5. Internal volume within the inlet column joint including the porous sieve plate and flow channel.
6. Volume of the chromatographic column.
7. Internal volume inside the bottom column fitting including the porous sieve plate and flow channel.
8. Volume of the tubing from the column outlet to the detector inlet.
9. Volume of tubing inside the detector prior to the flow cell.
10. Flow cell volume.
The variance of all these volumes is cumulative. Thus, it is possible to reduce the injection volume to a degree and keep the length of the column inlet tube short and the inner diameter small, but if the connecting tube from the column outlet to the detector is 1 m, the band spread may be large enough to affect the overall efficiency of the separation. Therefore, if you wish to obtain the expected efficiency of a 1.8 μm HPLC column or a column with an internal diameter of 1.0 mm, ensure that out-of-column effects are kept to a minimum.
2) For silica fillers, residual silanols cause trailing peaks
In some cases, silanols present on all silica-based bonded-phase columns can cause tailing, particularly for basic compounds. This tailing is attributed to the fact that the silanol group is a weak acid with a pKa of approximately 4.5-4.7. Therefore, as the pH of the mobile phase approaches 4-5, silanols are ionised and can interact with positively charged molecules (e.g. protonated amines) by electrostatic attraction. This interaction can be largely reduced by lowering the pH to 3 to inhibit the ionisation of silanols. For some alkaline compounds, tailing can sometimes occur at lower pH values. There are specially designed reversed-phase packings that can provide good peak shapes for basic compounds.
There are three basic causes of peak dragging: chemical problems (one of which has been discussed earlier); column packing problems and instrument hardware problems. All three of these factors can contribute to peak tailing and the underlying causes must be investigated to determine remedial measures. It is beyond the scope of this issue to discuss these three problems in detail, but some examples of each type will provide you with general ideas. First, chemistry-related tailing problems can manifest themselves in several ways, in addition to interactions with silanols. Metal chelating compounds can interact with trace amounts of metal in the column filler, which is particularly noticeable with older silica materials.
Using the wrong injection solvent can sometimes lead to trailing. Injection of a sample with a much stronger solvent than the mobile phase can produce distorted peak shapes. Trailing can be caused by a build-up of material caused by strongly retained sample components or mobile phase impurities at the top of the column. This material can act as a different stationary phase, which can lead to dragging problems with interacting compounds. Sometimes a mixing pattern can occur, which can lead to trailing. Here, the solute can interact with the active group through reversed-phase and ionic interactions. Sometimes, when the pH of the mobile phase is wrong and the sample is partially ionised, the resulting peak can be distorted and resemble a drag-tail. The partially co-eluting peak on the back of the larger peak is smaller and sometimes looks like a drag.
Column-filling problems can lead to trailing peaks. Gaps at the top of the column can lead to double peaks or trailing peaks. Inadequately packed columns may form channels, leading to poor peak shape. Trailing may occur if the column is overloaded due to excessive sample mass, although peak fronts are more common. Trailing may occur with silanol overloading at low feed masses.
Let’s consider some of the instrument and hardware issues that can cause tailing. Trailing may be caused by off-column effects and other uncleared volumes in the flow path. Uncleared volumes caused by incorrect end connections or connections may show up as peak trailing. Pressure pulses from the injector may cause column voids, which can lead to trailing. Slow detector time constants and fast eluting summits cause peak broadening, sometimes similar to trailing. A thermal mismatch between the solvent entering at room temperature and the higher column operating temperature can also lead to peak distortion. Therefore, it is not always the silanols that cause peak trailing in HPLC.
3) Silica gel fillers can only be used in the pH range of 2-7
Typically, there are two mechanisms of chemical degradation of HPLC reversed-phase columns: catalytic hydrolysis of siloxane bonds at pH less than 2; and liquid chromatography. Hydroxide ions at pH greater than 7-8 can solubilize silica. At pH values less than 2, the -Si-O-Si- (siloxane) bond can be attacked by a hydrogen bond (H3O +) cleavage of the bonded phase, resulting in a loss in organic. With the passage of time, retention usually decreases with decreasing carbon content. This is particularly noteworthy for short-chain capped portions, such as trimethylsilanes. The longer C18 phase offers some protection to the siloxane below due to its spatial site resistance, but can eventually even be subject to erosion, especially at temperatures above the ambient temperature. Having a spatially protected and tightly covered bonding phase can help to stop the cleavage of the bonding phase on the silicone. Polymer phases do not show this instability but have the disadvantage of being less efficient than silica-based phases.
In terms of high pH, there must be some way to stop the silica below being attacked by hydroxide ions. Once the dissolution process has taken place, the column will eventually fail and voids will often form. Special columns designed for high pH operation include diphthong C18, hybrid, and polymer-coated silica. These packings are able to withstand pH values in the range of 11-12. When operating in this range, most silica-based fillers should be protected from high temperatures. Of course, polymer phases can operate at pH values up to 13 and sometimes even 14, but they have the disadvantage of being less efficient than silica-based phases.
The real answer, therefore, is that special silica-bonded phases can be operated outside the pH range of 2-7, but care should be taken to use conventional silica fillers, especially at high temperatures.
4) Modern HPLC columns should withstand at least 1000 feeds
There are a number of factors that control the number of feeds that any modern HPLC column can withstand. Some factors are based on mode: reversed phase, ion exchange, volume exclusion, normal phase, chirality, hydrophilic interactions, etc.
Some of these factors depend on the type of packing in the column, such as silica packing, hybrid packing, zirconia packing, or polymer packing, or the degree of cross-linking of soft gels or resins. Other factors depend on the operating conditions: pH, temperature, mobile phase composition, buffer composition, flow rate, pressure, etc. It also depends on the sample injected: standard sample only, sample cleanliness, sample pH, sample volume, impurities present in the sample, solute molecules themselves, etc.
If the column is abused, for example by using it outside its recommended pH limit or flow rate range, only 50 injections or less may be made. If the sample is simple and there are no highly retained impurities, the column can withstand 5000 or more injections. The column will last longer if it is not run continuously at its upper limit.
Most people expect a minimum of 1000 feeds when a 5 μm reversed-phase column is used to analyze formulations, simple drug mixtures, and standards. If the column is used for “dirty samples”, such as biological liquid extracts or environmental extracts that have not been completely removed, then 1000 injections should not be expected.
The expected number of injections is therefore not fixed but depends on the type of column, operating conditions, sample cleanliness, and level of abuse. Of course, for the use of protected columns and in-line filters, the column life should be longer. Many chromatographers do not actually know how many injections can be obtained per column life. Some modern HPLC instruments have built-in column modules and software that enable them to monitor the number of feeds.
5) The column should always be tightly capped to prevent damage to the packing from coming into contact with the atmosphere
Typically, the small holes in the end connections, which may be no larger than 0.02″ in diameter, have such a small cross-sectional area that air entering the packing and solvent contact inside the solvent can damage the column. Evaporation from the column is minimal. Even if a small amount of air enters the column, it may be difficult to diffuse through the particulate-packed bed and come into contact with sufficient packing material to have an adverse effect. If a small amount of air is present in the column, the small amount may be dissolved or flushed out under high pressure when it is pressurized immediately after it is next installed in the HPLC instrument and the mobile phase is pumped to it. The initial liquid will not cause any problems for subsequent use for a short period of time. However, if it is essential to feel safer by sealing the end connections, then do so. Most columns are equipped with column connectors and these screw caps can be tightened to prevent any possibility of evaporation of the storage solvent or air entering the packed bed.
