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Ten Misunderstandings in the Use of Liquid Chromatography Columns


1. Can’t be reverse use of HPLC columns


Wrong! In fact, the packing pressure of the HPLC column is much higher than the maximum use pressure (usually twice ). A well-packed column can be used in both directions if using proper homogenate agent and distribute a certain amount of time to stabilize the column bed. The reverse use of chromatography columns includes: backwash during column conversion, column head with strong retention substance adsorption, contaminatable chromatography column, (The backwash path is shorter and more reasonable), and washing the particles remaining on the frit to reduce the column pressure or prevent the column pressure from rising.

An exception to the reverse use of chromatography columns is the use of larger frit at the injection end of the column by the manufacturer, which may flush the packings out of the column bed. When the chromatography column is packed in the factory, the outlet-end frit aperture must be smaller than the particle diameter of the smallest particle in the chromatography column. For example, the average particle diameter of chromatography packing is 5μm, the particle diameter distribution range is 3-7μm, and the outlet-end frit aperture must be less than 3μm, which makes packing impossible to run from the column bed to the outside of the chromatography column frit. The most manufacturers choose 2μm.frit aperture.

Q: Why do some chromatography column manufacturers choose the different frit apertures at the inlet and outlet ends?

A: Simply, the large frit aperture is not easily blocked compared with the small frit aperture. A 0.5μm diameter frit will be blocked faster than a 2μm diameter frit. So in order to avoid the rapid rise of column pressure and customer complaints, manufacturers will compromise to use a frit with a slightly larger aperture at the inlet end. Usually an arrow will be marked on the column to indicate that it can only be used in one direction. When reversely using HPLC column, it is better to look up the instructions carefully or consult the column manufacturer whether can be used reversely.


Note: Before the column is washed in the opposite direction, the column must be connected back and do not connect the detector for a period of time. In case of impurities blocking the flow cell. Generally, normal chromatography columns should be used reversely after washing in the opposite direction.


2.All C18 (L1) columns are the same


Wrong! In the early development of HPLC, octadecyl silane (often called ODS or C18) was one of the earliest bonded stationary phases, which also led to the birth of "reversed-phase chromatography" new technology. C18 has become the standard stationary phase for reversed-phase chromatography and has been adopted by most chromatographers.

Pharmaceutical industry is the earliest user of HPLC technology. Regulators do not want to favor the brand names of any chromatography column manufacturer. FDA and USP have compiled a classification system to give a generic name to each new method of drug application. For the HPLC column, it is named "L1" C18, because it appears in most of the materials submitted for examination, so it naturally becomes "L1". With the increase of other stationary phases, each has its own "L sequence number" (e.g.:"L7 is C8, L10 is CN, L11 is phenyl, etc.). Unfortunately, this naming system has proved to be unreliable. Because each commercialized C18 column, although silica gel is also used as the matrix, has its own specific packing bonding synthesis process, so the chromatographic performance is different. For example, some manufacturers use octadecyl monochloramine bonding reagent and low surface area silica gel (see figure 1), while others use the same silane reagent but choose higher surface area silica gel matrix. The chromatographic performance of these two C18 columns is different, and the bonding ratio of the latter C18 stationary phase is larger than that of the former.


3.Guard column does not affect separation effect


Wrong! First of all, it's a good idea to have a guard column. If the type of guard column or the selection of stationary phase is wrong, the guard column can also have a great impact on the separation effect. It should be made clear that the guard column is used to protect the analytical column from contamination by strong retention components, particulates and other unnecessary materials. Guard column is much cheaper than analytical column, so polluted guard column is replaced more frequently. Ideally, the stationary phase of the guard column is exactly the same as that of the analytical column. If the stationary phase is retained strongly (such as high carbon loading and mixed phase), it may even lead to different selectivity. If the retention is weak, the problem is not too obvious unless the stationary phase affects the selectivity of the whole system. In order to minimize the separation effect of the guard column, the guard column needs to be properly assembled. Obviously, the guard column is inserted between the sample inlet and the analysis column, but if the pipeline is too long (too long or too big inside diameter), it will cause additional peak broadening, which will affect the separation.

The integrated guard column (the guard column is directly connected to the chromatography column) was introduced into the system. From the previous reports, it can basically reach the optimum level of the analytical column. However, integrated guard columns are usually used with card-type chromatography columns, which are not so popular at present. No matter what type of guard column is used, it should be easily removed and replaced from the HPLC system without affecting the operation. In theory, the length of analytical column is prolonged by attaching guard column, which will increase column efficiency and chromatographic separation effect. But, due to the influence of some factors discussed above, adding guard column can only maintain the original chromatographic separation performance at the best level, sometimes even worse. The advantage of using guard column is to prolong the column life, not to improve the overall chromatographic separation effect.


4. Increasing the temperature is often beneficial to the separation effect


Wrong! With the increase of temperature, the viscosity of mobile phase decreases, so the transfer rate of analyte increases, so it can provide better chromatographic separation effect. In addition to column efficiency, temperature also affects retention factor (k) and selectivity (α). Temperature variations can improve or decrease resolution (which is also the most important concern of chromatographic analysis). The retention time often decreases with the increase of temperature, because temperature is a thermodynamic parameter, which makes the analyte tend to stay in the mobile phase at high temperature, and it can be eluted from the column more quickly. Then, different compounds may have different retention time in response to temperature changes. More precisely, their Van 't Hoff lines have different slopes (lnk and 1/T, T measured at absolute temperature); in other words, the value of alpha changes. In addition, the increase of temperature will make the peak of low k value chromatographic peak come out faster, even near the unreserved substance t0, which makes it difficult to quantitative analysis.

Analgesics (a kind of analgesic drug, translator's note) as an example, the second figure list a series of chromatography figures that show the separation of seven kinds of analgesics in the column temperature with 20-90°C. We can find many characteristics from it. Firstly, with the increase of temperature, the retention time of all analytes becomes shorter and the peak becomes narrower, which means that higher temperature is beneficial to the separation effect. Secondly, one of the analgesics, salicylic acid (aspirin) is between peak 5 and peak 6, and its position changes greatly with the change of temperature. The fact is that the elution sequence of the substance changes with temperature change at 20-40°C. Salicylic acid peaked with the 6th phenacetin in the middle 30°C. Therefore, the separation time and elution sequence will change when the temperature is higher than 40°C. Of course, one advantage of increasing temperature is that the operation column pressure is reduced to facilitate the use of larger flow rates and smaller packings. Another factor that can cause performance problems of chromatography column at high temperature is the temperature difference of mobile phase. If the column operates at 60°C, but the mobile phase flows into the column from room temperature condition, then the cold mobile phase can cause peak deformation, because the analyte tends to be in the high temperature mobile phase at different temperatures. Therefore, it is suggested that the mobile phase should be preheated when using chromatography column at higher temperature.


5. The higher the carbon loading is, the better the reversed-phase chromatography column will be


Wrong! When it comes to ordinary alkyl bonded phases, such a conclusion seems to misunderstand the concepts of chain length, carbon-loading amount and surface bonding degree. Usually, for a true reversed-phase retention mechanism, the retention ability depends on the relative hydrophobicity of the molecule being analyzed, so the retention generally depends on the amount of carbon loading. The higher the carbon-loading amount is , the stronger the retention capacity will be. Carbon-loading is amount generally proportional to chain length, but not necessarily.

Typical chromatographic silica gel surface, silanol content of about 8.0μmol/m2 can be used to bond with organosilane reagents. Assuming that single-layer bonding can be achieved on all silanol groups, the longer the chain length is, the larger the carbon-loading amount, and the result is that the retention capacity is proportional to the chain length. Short-chain bonded phases (e.g. C8), if the surface bonding degree is relatively higher, then bonded carbon will be more, which may lead to stronger retention than C18.

In addition, some manufacturers use dichlorosilane and trichlorosilane as bonding reagents, because the occurrence of polymerization can generally increase the bonding rate of stationary phase. In this case, the bonding degree of short chain polymer bonded phase is much higher than that of long chain monomer bonded phase. Compared with monomer bonded phase, the bonding layer of polymer bonded phase is thicker, which sometimes causes the mass transfer rate to slow down. The reversed-phase column with high carbon loading is more prone to phase collapse. For these chromatography columns, when the proportion of organic phase in mobile phase is below 10%, the hydrophobic stationary phases tend to self-associate with each other rather than dissolve in polar aqueous solution (i.e., similar phase dissolution). Therefore, high carbon loading may be harmful to the performance of reversed-phase chromatography, and the reproducibility of retention time is not good.


6. Chromatography column with small particle diameter must have better separation effect


Wrong! For a well-packed chromatography column, the column efficiency increases with the decrease of the average packing particle diameter, but for a specific HPLC instrument, if the Extracolumn effect causing peak broadening is large enough, the high column efficiency of the small-particle chromatography column can not be achieved. Extracolumn peak broadening effects are caused by all additional volumes other than the packing itself, which include:

a. Sample volume, including additional volume in the injection ring and valve

b. Pipeline volume from the outlet of the injection valve to the inlet of the chromatography column

c. Clearance volume in the guard column (if the guard column has a column head, it also includes the volume in the head)

d. Volume in online filter

The above mentioned volumes are all Extracolumn volumes. Therefore, even if the injection quantity can be minimized, the pipeline before the inlet of the column should be kept as short as possible and the inner diameter should be as small as possible. However, if the 0.2 inch inner diameter pipeline is 1 meter long, the spectrum band broadening effect may greatly affect the overall effect of chromatography separation. Therefore, in order to achieve the desired separation efficiency on a column with a packing diameter less than 2μm or a microporous column with 1.0mm inner diameter, it is necessary to ensure that the Extracolumn effect of the HPLC system is minimized.


7. For silica gel packings, the residual silanol group is the cause of tailing


Wrong! Under specific conditions, silanol groups exist in silica gel matrix bonded columns, especially in the analysis of alkaline substances, can cause peak tailing. The reason for the tailing is that the silanol group itself is a weak acid, with pKa between 4.5 and 4.7. Therefore, if the mobile phase pH is between 4 and 5, silanol will ionize and interact with positively charged molecules such as protonated amines through electrostatic attraction. This effect can be minimized by reducing the pH below 3 to inhibit the ionization of the silanol group. But for many alkaline compounds, tailing can also occur at low pH. With specially designed reversed-phase chromatographic fillers, good peak shape can be obtained for the determination of alkaline compounds.

Three reasons can cause tailing, chemical problems, column packing problems and chromatographic equipment hardware problems. Three kinds of problems can cause tailing, but to solve the problem, we need to find the root cause of the problem. It is beyond the scope of this article to explore these three reasons in detail. Here, we give several examples of the three types of problems to give you a general understanding.

First of all, In addition to the role of silanol groups, there are several other ways of tailing chemical factors. The tailing caused by chelating compounds with metals and trace metals in chromatography packings is particularly evident in early silica gel substrates. Incorrect solvent selection can lead to tailing, and the use of stronger solvent injection than mobile phase can also lead to peak distortion. The accumulation of strong retention components and impurities in mobile phases in the column, which are not easy to be eluted in the sample, can also lead to tailing. These substances accumulated in the column can play different roles as stationary phases and interact with the analyte through which the peak shape of the analyte is tailed. Sometimes, the retention of mixed modes can also induce tailing, such as the interaction between the analyte and the active group. Sometimes, incorrect pH selection of mobile phase, partial ionization of sample, coexistence of molecular and ionic states, and mixed mode interaction can cause peak distortion and tailing. In addition, a small peak behind a large peak appears to be the same as a trailing peak.

Columns with poor packing quality can also cause tailing. The packing clearance of the column head can cause peak bifurcation or tailing. If the column is not packed well, a small groove on the column bed can make the peak shape too wide. When the sample concentration is too high, the sample overload will also cause tailing, and the peak delay caused by sample overload is more common. If the sample is fewer, because too much silanol group exists, it can also cause tailing.

Next, we will discuss the tailing caused by the hardware problems of the instrument. The pipeline dead volume and joint volume mentioned above can cause tailing. Instantaneous pressure during injection can cause packing collapse (void formation) and tailing. When slower detector is used to detect rapidly eluted analytes, peak broadening and tailing will occur (which are related to the thickness and length of the flow cell). The inconsistency between column operating temperature and mobile phase temperature mentioned above can also cause tailing. Therefore, it is not always silanol group that causes HPLC tailing.


8. Silica gel packings can only be used at pH 2 to 7


Wrong! There are usually two mechanisms for chemical destruction of reversed-phase columns: catalytic hydrolysis of silica-oxygen bonds below pH 2 and dissolution of silica gel by OH-ions in water above pH 7 or 8.

When the pH is less than 2, -Si-O-Si-can be attacked by the hydrated hydrogen ion H3O+, and the stationary phase will lose and break. With the passage of time, the retention value will gradually decrease with the decrease of carbon loading. The phenomena are more obvious in short-chain-terminated chromatography columns such as trimethylchlorosilane because the end-capping reagents are more easily hydrolyzed and lost. Long-chain C18 stationary phase has some protective effect on such loss due to steric hindrance effect, but it is still slowly eroded, especially when the temperature is higher than the environment. Space protection and high density bonding are helpful to prevent the loss of silica gel bonding phase. Polymerized stationary phase performs well under these conditions, but its column efficiency is lower than that of silica gel stationary phase. In the aspect of high pH, it is necessary to take measures to protect silica gel matrix from attacked by hydroxide ions. Once the dissolution process begins, with the appearance of voids in the column bed, the chromatography column will eventually fail. Therefore, special chromatography columns with high pH resistance, such as bidentate C18, organic-inorganic hybrid particles and polymer-coated silica gel, have been developed. These columns use chemical methods to protect the stationary phase from OH attack. These special columns can withstand the conditions of pH 11-12. In this alkaline pH condition, the column should avoid high temperature use. Of course, polymer columns can be used at pH 13 or even 14, but it has been pointed out that polymer columns are less effective than silica gel columns.

Therefore, in this argument, special silica gel columns can be used outside the range of pH2-7, but ordinary silica gel columns should be very careful, especially at high temperatures.


9. Contemporary HPLC columns should withstand at least 1000 injections


Wrong! The number of injections that modern HPLC columns can withstand is determined by many factors. Some factors vary according to the following chromatographic modes: reversed phase chromatography, ion exchange chromatography, exclusion chromatography, normal phase chromatography, chiral chromatography, hydrophilic interaction chromatography, etc. Some factors are based on different packing substrates: silica substrate, hybrid substrate, zirconia substrate, polymer substrate, or gel and a certain degree of crosslinking polymer resin. Some factors are based on the properties of the stationary phase itself: surface bonding degree, bonding mode, polymerization or monolayer bonding, or polymer coating. Other factors are related to chromatographic application conditions: pH, temperature, mobile phase composition, buffer composition, flow rate, pressure and so on. Others are related to the sample: complete standard sample, sample cleanliness, sample pH, sample volume (content), sample impurities, molecular characteristics of the analyte, etc. If a column is abused, such as outside the range of pH or velocity, it is unlikely that withstanding 50 times injections. If there is no strong retaining substance in the sample, it is also possible to withstand 5000 times injections. If the chromatography column is not continuously used at the upper limit of its bearing capacity, its life will be longer. If the sample injected into the column is complex, but the strong retained substance in the column is never washed out, the life will inevitably decrease.

According to the experience gained through interviews with many pharmaceutical companies, showing that most of 5μm reversed-phase chromatography columns can withstand at least 1,000 injections in the case of analytical preparations, simple drug mixtures and standard samples. If the samples are "dirty", such as the extracts of biological samples and environmental samples, which have not been strictly purified, it can not reach 1000 injections.

So the times of a column can withstand is not absolute, but depends on the type of column, operating conditions, sample cleanliness and abuse of the column. Of course, using guard columns or online filters will prolong the life of the chromatography columns. In many professional seminars, the author conducted informal polls among the audience. The results showed that less than 15% of the users actually recorded the times of a particular column could be used. Many chromatographers did not really know how many times each column could be used.


10. Chromatography columns should always be tightly sealed to prevent packings from being damaged by air contact



Wrong! Usually, the diameter of the micropore at both ends of the column is less than 0.2 inches, so in such a small cross-sectional area, the damage caused by air entering into contact with packings and solvent evaporation in the column is very small. Even if part of the air enters the column, it is difficult to diffuse through the compact packed bed and contact enough packings to cause damage. If there is some air in the column, as long as it is running once on the chromatography equipment, it will dissolve immediately under high pressure, and then flow out. It will not do any harm to the later use. Of course, if you want to insure, you can tighten threaded plunger heads. Most of the columns are equipped with threaded plunger heads, which can be tightened manually to prevent solvent evaporation and air entering into the packings.