3b01

The main chemical difference between the stationary phase and the mobile phase in reversed-phase HPLC can be described in various ways: we say that the water/organic phase is more polar while the alkyl bonded phase is less polar. We can describe the mobile phase as hydrophilic (water-loving) while the bonded phase is hydrophobic (water-hating). The bonded phase is somewhat like a wax or oil layer, repelling water and compounds that dissolve well in water.

Compounds that are more polar prefer the polar mobile phase and move through the column more quickly. Compounds that are non-polar tend to prefer the non-polar bonded phase and move through the column more slowly. Looking at the separation on the right, we see that retention increases as we increase the non-polarity of the compounds (nitrobenzene is the most polar of the these compounds; adding alkyl substitution decreases the overall polarity).

Separation of nitroaromatic compounds on a C18 column with a 60% methanol/water mobile phase

Generally, a decrease in water content of 10% (e.g., going from 60% methanol/water to 70% methanol/water) results in a 2- to 3-fold decrease in sample retention time. Therefore, changing the water content of the mobile phase is a good way to control the sample separation time. Notice, however, that decreasing the water content in order to shorten the separation time also generally makes the separation worse; the peaks move closer together and are not as well resolved.

Separation of the same sample shown above using 70% methanol/water mobile phase

There are about ten solvents that are used for HPLC mobile phases. These solvents must be specially purified before use: so-called HPLC grade solvents. Water is an especially critical solvent, and we use a lot of it. For this reason, many labs purify their own HPLC-grade water using special systems from companies such as Millipore or Barnstead.

In reversed-phase chromatography (the most commonly used technique), we usually use only 3 organic solvents for the mobile phase: methanol, acetonitrile, and tetrahydrofuran (THF). These are the three solvents that are the most different from one another in their chromatographic selectivity and fully miscible with water in all proportions. Solvent miscibility is quite important, because an LC system will not work with a solvent combination that is immiscible. The common reversed-phase solvents, water, methanol, acetonitrile and THF are each miscible with each other. You can blend them together in any proportion, and use any combination of 2 to 4 of these solvents. Combinations of two solvents (usually one organic solvent plus water) are referred to as "binary" mobile phases. Combinations of three solvents (two organic solvents plus water) are referred to as "ternary" mobile phases.

A critical solvent property is the UV cutoff wavelength. This is particularly important when using a UV detector - which is generally the case in LC. This UV cutoff wavelength indicates the lowest wavelength that a UV detector can be used with that particular solvent. If a lower wavelength setting is selected, no light gets through the flow cell (it is all absorbed by the solvent), and the detector will not work properly. You will probably find that the signal is pegged to the top or the bottom of the display, regardless of what bands are passing through the flow cell. For example, the UV cutoff for methanol is 205 nm. This means that the detector setting will have to be greater than 205 nm, and probably at least 210 nm.

UV Cutoffs of Useful HPLC Solvents

Solvent	Cutoff
	(nm)
hexane	195
n-butyl chloride	220
methylene chloride	233
chloroform	245
methyl-t-butyl ether	210
tetrahydrofuran	212
ethyl acetate	256
acetonitrile	190
methanol	205
water	190

Another important solvent property is its viscosity. Solvents that have greater viscosities are harder to pump through the column, and cause proportionately higher pressure readings. So if you change from a solvent like methanol (viscosity at 250°C equal 0.55 centiPoise, cP) to a higher viscosity solvent like water (viscosity = 0.89 cP), you can expect the pressure to go up by the factor (0.89/0.55) or about 1.6-fold. Often a mixture of two solvents will have a viscosity that is between the viscosities of each solvent. However in reversed-phase LC this is often not the case. The viscosity of a 60% methanol/water mobile phase, for example, will be 3-times greater than for pure methanol, and 50% higher than for pure water! This means that the pressure when pumping this mixed mobile phase will be signficicantly higher than it would be when pumping either of the pure components.

Now let's consider how we make up the mobile phase. Usually a mixture of two solvents is described in terms of volume. For example, 30/70 methanol/water refers to 30 parts-by-volume of methanol added to 70 parts-by-volume of water. We could obtain this mobile phase mixture by pouring out 300 mL of methanol in one graduated cylinder and 700 mL of water in another. Then mixing these two solvents will give about a liter (1000 mL) of 30/70 methanol/water. The final volume will not be exactly 1000 mL in this case, so it would NOT be correct to simply fill the first graduated cylinder (containing 300 mL of methanol) to mark (1000 mL) with water. This same mobile phase can also be called 30%v methanol/water.

When the mobile phase contains added buffer, ion-pair reagent or other added ingredients, it is customary to weigh out these substances, add them to the aqueous portion of the mobile phase mixture, then add the organic. For example, if we want to make 2000 mL of 40%v methanol/20 millimolar potassium phosphate at pH 2.7, proceed as follows:

First calculate the weight of potassium dihydrogen phosphate required for 1000 mL at 0.02M concentration (20 millmoles). Dissolve the salt in the water.
Next calculate the weight of potassium hydrogen phosphate required for 1000 mL at 0.02M concentration. This is also 20 millimoles. Dissolve the salt in the water.
Now mix the two phosphate solutions as required to obtain the desired pH (do *not* put the pH electrode into the bulk solution to measure pH; instead, remove aliquots of the solution and check pH "off line", then discard the aliquots. This minimizes the possibility of contaminating your mobile phase with buffer from the electrode.).
Finally, measure out 1200 mL of the buffer solution and add 800 mL of methanol.

If you are running a method which specifies a different procedure for mobile phase preparation, be sure to follow the procedure exactly as written. Even small changes in mobile phase preparation can have a significant effect on your chromatography.

Before using the mobile phase, it should usually be degassed. This is not always required, but many LC pumps and detectors work better when the mobile phase is free of dissolved air. Degassing the mobile phase was discussed in detail in Section 2.

The mobile phase has a significant effect on the pressure of the LC system, as we discussed above. The pressure also depends on column length and flow rate. Finally, the particle diameter of the column packing has an important effect on pressure: smaller particles give higher pressures.

When it comes to measuring pressure, different LC systems often use different units. This can be confusing when we try to compare pressures between different systems. Many systems use pressure units of ATMOSPHERES (also called BARS), others use MEGAPASCALS (MPa), while still other systems use pounds per square inch (PSI). If you want an approximate way to interrelate pressures in different units, one bar is equal to about 15 psi or 0.1 MPa.

We should keep an eye on the system pressure, which will usually be displayed on a gauge or other readout. A gradual increase in pressure over a long time is common for most columns, but sudden increases in pressure - without any reason - are cause for alarm. Also the pressure should remain fairly constant from one minute to the next, without any sudden surges or random oscillations. The LC system cannot tolerate pressures above a certain limit, which is usually 4000 to 6000 psi (250-400 bar or 25-40 MPa). Most LC pumps today come with automatic shut-off controls if the system pressure rises above some critical limit. Even though the pump might tolerate higher pressures (e.g., 8000 psi), this pressure shut-off limit can be set at a much lower level (e.g., 2000 psi).