Why hplc high pressure

If a gradient method is used, record the pressures under the starting conditions. You may want to shortcut the process and just record the pressure with all components installed, the column inlet disconnected, and the in-line filter if used disconnected; this approach will isolate the most common sources of system blockage for future reference.

Because method pressure rises normally over time as frits and filters collect debris, I like to track the pressure. A convenient way to do this is to add a "starting pressure" item to the data recorded at the beginning of each batch of samples column serial number, sample batch number, notebook reference, and so forth. These data can be used for future reference or plotted over time as a control chart to help anticipate pressure problems before they occur.

You may want to have an estimate of what the normal system pressure should be, just as a cross-check. The technique I like for this uses equation 2. For pressure in bar, divide by The mobile phase viscosity will depend on the components in the mobile phase and the temperature. Methanol and acetonitrile are the most common organic mobile phase components for reversed-phase LC, mixed with water or buffer. I have summarized the viscosities of mixtures of methanol and acetonitrile with water at several compositions and temperatures in Table I.

For a more complete listing, see Table 1. Table I: Viscosity of mixtures of methanol or acetonitrile with water. Data from Table 1. Now we can use equation 1 to estimate the pressure for a selected method. Several examples are given in Table II. This is because the resistance to flow of some columns may differ because of packing techniques, and the quoted nominal particle size may not be the true value.

For example, a 0. I have included the pressure calculated for the above example and several other common column configurations in Table II; in each case the columns are selected to give approximately the same plate number, N , so a similar separation should be obtained in each case assuming identical column chemistry. A few general observations are in order. For comparison, I have also included a shell-type particle.

The particle size 2. The right-hand column of Table II lists the column dead-time, t 0 , which can be used to compare run times for the various columns. Also, a 1. And finally, the shell-type 2. Now that we have a technique to approximate the column pressure, we can see how calculated values compare with the observed values under the system reference or method reference conditions. If you are using a UHPLC system, you'll need to add to the calculated value the system pressure observed when the column is removed, which may be — psi.

A gradual increase in pressure over time is a normal symptom of column aging, and excessive pressure is often the first indicator that something is wrong with the system. In some cases, the pressure increase may be large enough to trigger the upper-pressure limit, and system shutdown may occur. High pressure is a symptom that something in the flow path is partly or completely blocked.

The most common location for this will be the first frit after the autosampler because it accumulates debris from the sample or other sources.

This is one of the reasons I strongly recommend using an in-line frit just downstream from the autosampler. Use a 0. This frit has smaller porosity than the frit at the head of the guard column or column, so it will become blocked first.

The frit in the in-line filter is easy and inexpensive to change, making it a quick fix for the most common high-pressure problems and a simple way to protect the expensive column from damage. Isolate the location of the blockage by progressively loosening fittings, as described earlier, until you find the source of the pressure increase.

These consumable items are subject to day-in and day-out mechanical stress and eventually wear out. The devices contain polymeric sealing materials that are subject to the same wear problems as injector rotor seals.

The particulates can be small enough so as to pass through some of the HPLC system components for example, mixers, pulse dampers, and tubing but large enough to lodge in a porous frit at the inlet to the column. An overlooked source of particulates is salt precipitates. Aqueous buffers often are used in reversed-phase, ion-exchange, and aqueous size-exclusion chromatography.

If the aqueous—organic mobile phases are created in the gradient pumping system, at high organic levels there might be the possibility of precipitation or emulsion formation upon mixing. It is best to externally check the highest organic solvent composition added to the aqueous solution to make sure that no cloudiness occurs indicative of salt precipitation.

Such a check for precipitants also can prevent seal wear within the flow system. Also, do not forget that the sample solvent can be a source of salt precipitation. If the sample is loaded with salt and it is injected into a high-organic mobile phase, the salt in the sample can precipitate.

In this case, a simple desalting procedure can be used to remove this salt. Vice versa, if the mobile phase is high in salt and a large volume of organic solvent is used for the sample injection, salt in the mobile phase can come out of solution.

The best way to deal with particulates is to remove them before they reach the inlet frit. Samples and even standards should be filtered using HPLC membrane syringe filters, available from a variety of manufacturers. Clean liquid samples prevent the blocking of capillaries, frits, and the column inlet. In addition, clean samples result in less wear and tear on the critical moving parts of injection valves. Sample filters come in a number of different porosities and diameters.

For use with 3. If sample volume is limited, use the smallest possible filter diameter such as 13 or 25 mm.

An important consideration is the choice of membrane. Choice of membrane is dictated by injection-solvent and analyte compatibility with the polymeric material. Syringe filters made from materials such as regenerated cellulose, PTFE, nylon, cellulose nitrate, and cellulose acetate are common in the HPLC laboratory.

Readers are directed to syringe-filter manufacturers' literature that often have solvent compatibility tables or to an excellent article in a previous issue of LCGC For mobile phases, especially self-made buffers, filtration also is recommended. Clean mobile phases reduce wear on instrument parts for example, check valves, piston seals, and autosamplers. Again, make sure that the filter membrane used is compatible with the mobile phase solvent being filtered.

Simple vacuum filtration flask systems are available commercially. An added benefit is that in vacuum filtration, the mobile phase is degassed also but most modern HPLC systems have in-line degassing as a standard feature. Mobile phase filters also can be placed in the solvent reservoir.

It is attached to the end of the reservoir tube between it and the pump inlet. Otherwise, cavitation can occur. A popular approach to ensure clean samples or mobile phases is to install an in-line filter see Figure 2. These days, in-line filters have minimal dead volume, so they provide minimal contribution to band broadening. The filter can be inserted between the injector and the analytical column column prefilter or between the pump and the injector.

When placed between the injector and the column, the filter protects the column from mobile phase as well as sample particulates. When placed between the pump and the injector, the filter protects against mobile phase particulates only. Some in-line filters have replaceable filter elements and are finger-tightened. For samples that are sensitive to stainless steel, metal-free in-line filters are available. In-line filters must be able to hold up to the column operating pressure because they are installed on the high-pressure side of the pump.

Figure 2: Schematic of an LC system showing in-line devices used to prevent back-pressure problems. Another particulate removal technique is the use of a guard column see Figure 2. Normally, a guard column is recommended to prevent chemical not mechanical contamination of the analytical column see the following. It is placed between the injector and the HPLC column. A guard column is a shorter version of the analytical column and normally should be packed with the same packing material.

Because the guard column frequently is constructed in a similar manner to the analytical column, it is the first system component that particulates might meet if no in-line filter is installed. Thus, it would become plugged before the analytical column is plugged. Because the cost of the guard column is a fraction of the analytical column, it is generally discarded and replaced when its pressure rises or it becomes contaminated.

Once the inlet filter becomes blocked and the column pressure rises unacceptably, there are a couple of approaches that can be tried to rectify the situation.

The most popular approach is to backflush the column by removing it from the chromatograph and attaching the inlet line from the injector to the column exit. The idea here is to dislodge accumulated particulates by flushing them out of the inlet frit.

Do not connect the column to the detector during backflushing to prevent accumulated particles from being flushed into the detector flow cell and perhaps causing problems there.

The column outlet formerly the inlet! The initial flow rate should be lower than the normally used flow rate but can be increased as the particulates are removed.

Sometimes, this procedure does not do the trick because the particulates have become embedded within the porous elements of the frit. One word of caution: make sure that the inlet frit's porosity is lower than the particle size distribution of the packing in the column as discussed earlier.

If such a column is backflushed, there is a chance of also flushing out some of the packing particles during the process. Check the column instruction or data sheet or call the manufacturer to make sure that their columns can be backflushed. If the column has an arrow on its side, this sign might be indicative of a column not to be backflushed.

In general, any well-packed column should be operable of flow in either direction. A second approach to solving a plugged inlet frit is to replace the endfitting. In general, I would not recommend doing this because the packing material that is in contact with the frit will almost always be disrupted because it sticks to the frit inlet fitting that is removed. Replacing this disrupted removed packing material with some additional packing will not provide a homogeneous bed, and column efficiency will be impaired.

Instead, if the backflushing cannot be accomplished or is not successful, it is probably best to discard the column. One other possibility of an increase in column pressure is a blocked flow line in the system between the pump outlet and the head of the column. A blocked line could result from a particulate lodging in the tiny internal diameter tubing that is now being used to decrease system dead volume required for the newer, high-efficiency columns.

A systematic removal of system components from the column backwards through the connecting line from the injector, any in-line filters or guard columns, the injector itself, and prior system components should eventually find the blocked element. Obviously, if the analytical column is removed from the system and the pressure readout is still high, the column is not the culprit so work backwards accordingly.

Be careful when performing this systematic removal at high pressure just in case a blockage is quickly dislodged because solvent can be sprayed from the component. So far, I have covered mechanical reasons for example, particulates or blockages why column pressure can increase. An equally strong possibility is pressure buildup caused by chemical contamination of the packed bed.

Just like mechanical contaminants, chemical contaminants can arise from a variety of sources — samples, sample matrices, mobile phases, or system components.

The most likely source of chemical contamination is from the sample, especially if insufficient or no sample preparation has been used to treat a complex sample. Usually there are compounds in the sample matrix that are of no interest to the analyst but are present nevertheless.

High molecular weight compounds, salts, lipids, waxes and fatty compounds, humic acids, hydrophobic proteins, and other biological compounds are but a few of the myriad possible substances that can come in contact with an HPLC column during its use. These materials can have lesser or greater retention than the analytes of interest. Those that have lesser retention such as salts usually will be eluted from the column at the void volume.

These undesired interferences might or might not be observed with the detector being used and, if detected, show up as chromatographic peaks, blobs, baseline upsets, sometimes even as negative peaks.

If sample matrix components are retained strongly on the column and if the mobile phase solvent composition itself never becomes strong enough to elute them, over the course of many injections, these adsorbed or absorbed compounds will accumulate, usually at the head of the column. Such behavior is observed more often when isocratic conditions are used. Sample compounds that are of intermediate retention can be eluted slowly and show up as wide peaks, baseline disturbances, or as baseline drift.

With gradient conditions, because the column is subjected to a stronger mobile phase during the run, sometimes these contaminants are removed in the latter stages of the gradient run. On occasion, the sorbed sample or mobile phase components build up to such high levels they can begin to act like a new stationary phase.

Analytes can interact with these impurities that now contribute to the separation mechanism. Retention times might shift, and tailing can occur. If sufficient contamination occurs, the column back pressure can build up to intolerably high levels, overpressuring the pump and perhaps causing the column to settle and void, depending upon where the blockage occurs.

It is this latter case that I will explore in this section. Noneluted sample components usually build up on the HPLC inlet column packing and, if unchecked, can cause these pressure buildups. The best way to remove chemical contaminants is to flush the column with a solvent that will dissolve the contaminants, yet will not harm the column packing itself.

For example, precipitated proteins often are removed from a polymeric column by flushing the column with strong base, perhaps of pH 13 or However, a silica gel-based column would most likely be harmed in this process, unless the column is a special reversed-phase column designed for high pH.

The best way to clean a column is to backflush not forward flush the column with a solvent or series of solvents that will remove the contaminants causing the pressure buildup.

Note that forward flushing is not recommended because sparingly soluble substances can take a long time to flush the entire length of the column and can become spread out during the process. Instead, in the reversed configuration, the pathlength from the inlet "chemical plug" to the column exit is much shorter and the washing time greatly reduced. Several years ago, I wrote an article on the cleaning and regeneration of reversed-phase HPLC columns In this column, I discussed approaching for washing silica-and polymeric-based reversed-phase columns to remove protein and other residues.

In addition, special techniques for cleaning reversed-phase columns when simple solvent washing does not work were covered. Rather than repeating the contents of the article here, I refer the reader to the original publication. Just remember, before washing any column used with a salt buffer, first remove the salt by flushing with a salt-free mobile phase of equivalent composition.

A similar article on the regeneration of biopolymer columns was published many years ago but is still applicable today For normal-phase columns such as silica gel, cyano, or diol, a similar procedure can be used in which progressively stronger solvents are used to wash contaminants from the column.

Isopropanol also can be used; it seems to have good solubility properties for fatty compounds. Be careful not to run too high of a flow rate during the washing experiments, as isopropanol's viscosity is quite high.

Microbial growth often occurs in buffers and aqueous mobile phases, especially if they are permitted to stand around for long periods of time at room temperature. Particulates or the bacteria from microbial growth can plug the column inlet or lodge on the column packing itself. Another overlooked source of chemical contamination is the use of the wrong rotor material in the injection valve for noncompatible solvents or pH. For example, Vespel, a frequently used polymeric material in rotary injector valves, is not compatible with high-pH buffers.

Prolonged use of such a valve under basic conditions will result in increased wear and earlier failure. Instead, in this case, a Tefzel valve core material is more compatible. Consult with the valve or instrument manufacturer on solvent compatibility of instrument parts.

The best way to prevent chemical contamination is to remove these undesired sample components before they reach the analytical column. Particularly creeping pressure changes can only be detected in case the pressure is monitored regularly.

The back pressure of the HPLC system should therefore be noted at regular intervals, at the latest each time the guard column is changed.

The use of a guard column is generally always recommendable, even when examining "clean" samples: The normal material abrasion of the HPLC pumps may be enough to impair the durability of columns. The best way to determine the back pressure is a standardized HPLC run, which is not changed.

In principle, you should always pay attention to the pressure, even - or especially - in routine measurements. To prevent pressure-related column or device damage, a maximum pressure should be set in the HPLC software, beyond which the pump switches off automatically. In this case, the pressure limit of the HPLC system and of the column used should be respected see the manufacturer's instructions.

Especially when you are routinely working, you may overlook the obvious. Thus, check at first if the system is properly connected are all capillaries connected, are there any leaks?

Is the mobile phase empty and may air have been sucked in? Leaks and air in the pump heads can account for a very low to no pressure. Another simple explanation can be a flow rate set too low, as well as a flow rate being too high will cause a too high pressure. Depending on the column dimension, there is a specific ideal flow rate that is best suited.

A flow rate being too high can damage the column. Especially during gradient runs, changes in pressure can occur, which can be explained by the viscosity of solvents and their mixtures. Gradients of water to methanol or tetrahydrofuran THF show a steep increase in pressure when mixed. Always make sure that the eluents used are miscible with each other! Determine the gradient-typical pressure changes by performing a run without sample injection and recording the pressure fluctuations.

Often, a baseline drift can also be seen which is quite normal for some gradients. In some cases, just after the sample injection, a spike in pressure is observed. With larger injection volumes, this can be normal.

The newly added volume at injection results in a short overpressure. In case it is a solvent of higher viscosity, the pressure can rise more than double for a brief period. If possible, use as sample solvent the same composition as the solvent used at the beginning of the run.

Inject the sample solvent "blank" to measure the pressure changes. If injection of a sample results in significantly higher-pressures values than the blank alone, the sample should be filtered or purified beforehand. We have just clarified pressure changes in gradient runs.

In gradient runs, the pressure can increase during the run and decline towards the end. Often a sinusoidal pattern of the baseline appears as concomitant. When raising the flow rate, the frequency of the pressure oscillation is also increased.