INSTRUMENTAL ANALYSIS AND SEPARATIONS

REVERSED PHASE CHROMATOGRAPHY - LECTURE 18

Reversed-Phase for Non-Ionic Analytes
Reversed-phase is the most common type of HPLC. Reversed-phase columns are
rugged, efficient, reproducible, and stable. Aqueous mobile phases are less expensive
than organic phases, detection is often easier, and less time is required for equilibration of
the column between runs. Reversed-phase chromatography is similar to the extraction of
compounds from water using an organic phase. The stationary phase, commonly C-8 or
C-18 is less polar than the water-organic mobile phase, and compounds partition between
the two phases based on their hydrophobicity. Polar (hydrophylic) molecules partition
less into the stationary phase and are eluted first. Non-polar molecules are more strongly
held by the stationary phase and are eluted last. Medium polarity molecules are eluted in
the middle of the chromatogram, and thus analytes can be separated.
If polarity was the only factor involved, compounds would always elute in the
same order. However, the retention of a compound is also affected by experimental
conditions such as, choice of the mobile phase, choice of stationary phase, and column
temperature.
Solvents for Reversed-Phase Chromatography
Partitioning between the mobile and stationary phases is based on solvent
properties collectively termed “solvent strength.” The three factors believed to be
involved in solvent strength are polarity, acidity, and basicity. A representation of the
strength of various solvents is presented in the solvent-selectivity triangle. It is possible
to completely reverse the elution order of some compounds by changing the solvent type.
Three water-miscible solvents, methanol, acetonitrile and tetrahydrofuran differ
considerably in their selectivity properties and are also acceptable in terms of UV
absorbance and viscosity and therefore are the most commonly used organic solvents for
reversed-phase separations. Methanol has the disadvantage of forming a relatively
viscous solution with water, giving high pressures, or requiring lower flow rates.
Acetonitrile is an expensive alternative to methanol. But in addition to offering different
selectivity, it does not produce the viscosity problems that methanol does.
Tetrahydrofuran offers additional selectivity, but its UV cutoff is relatively high (220
nm). Column re-equilibration following a gradient run is also much slower with
tetrahydrofuran than with the other solvents. Another disadvantages of tetrahydrofuran is
that it is unstable and when exposed to air it forms hydroperoxides that can react with the
sample, and may pose a safety hazard.
Mobile phases used should not contain less than 10% organic solvent when using
stationary phases such as C-18. When the mobile phase contains more than 10% organic
solvent, the alkane chains are more or less expanded out straight from the surface. With

Lecture 15

1

Most buffers (other than citrate) can be used for UV detection down to wavelengths of 200 nm or even less. and the pH is accurately measured. However. Compatibility with the detector is important. It is therefore important that the mobile phase is well buffered. For analyses conducted with a high pH mobile phase. Polar stationary phases such as amino. Lecture 15 2 . it can only be used with UV detectors at wavelengths above 230 nm. However. For most separations. Generally the longer the alkane chain. Stationary Phases Alkane chains of 8 (C-8) or 18 (C-18) carbon atoms bonded to silica are the most commonly used stationary phases. The other important solvent for reversed phase is water. because of its UV absorbance. extra care is needed to flush the system after a series of runs.higher water content. and diol can also be used in reversed phase chromatography. It is also important to note that the analyte itself can change the pH of the mobile phase within its concentration band so concentrations of the analytes should not be too large and the sample pH should not be too different from the buffer pH. This is because the citrate molecule has 3 ionizable groups with different pKa’s. It is also important that the buffer be compatible with the organic phase and not precipitate if high concentrations of organic solvent are needed for the separation.4). When the mobile phase pH is near the pKa of an analyte. cyano. Citrate has also been claimed to be corrosive to stainless steel. and trifluoroacetic acid can be removed by evaporation and are compatible with some detectors such as a light scattering detector or mass spectrometer. High purity water (HPLC grade) is needed to keep baseline noise to a minimum. the C-18 chains collapse and it is difficult to bring them back to the straight conformation when higher levels of organic solvent are added back to the mobile phase. so if it is used. alkanes bonded to a polymer support are used. In addition to the pKa there are other factors important to buffer selection.1 unit) and peak shapes can become skewed. the longer the retention. pH control is important so aqueous buffers are commonly used rather than pure water. ammonium acetate.1-6. ammonium formate. Buffers are limited in the pH range that they can control so it is best if the pKa of the analyte is similar to the pKa of the buffer. Volatile buffers such as ammonium carbonate. Currently available C-8 and C-18 columns differ significantly in their retention properties and are depicted in Figure 1. Citrate buffer has the advantage of being able to stabilize pH over a wide range (2. the retention of the analyte varies considerably with small changes in pH (as small as 0. retention is also based on the activity of residual silanol groups. Polymer supports are also preferred over silica for the analysis of basic compounds because the residual silanol groups on silica can cause severe band tailing (Newer type B silica can also be used for basic analytes).

Chromatography of Ionizable Compounds In reversed-phase chromatography. control of temperature can also be used to improve selectivity and resolution. Typically methanol or acetonitrile is used as the more polar solvent (weaker solvent for hydrophobic samples) while chloroform. a non-aqueous mobile phase can be used with what would typically be a reversed-phase column (C-8. methyl-t-butyl ether. Increasing temperature has the effect of decreasing k so that compounds elute earlier. but changes in temperature generally do not produce large changes in selectivity for non-ionic solutes. -carotene and lycopene is an example of a food science use of non-aqueous reversed-phase chromatography. However. sample retention increases for more hydrophobic compounds. For the separation of some carotenoids. long chains such as C-30 are needed to obtain adequate resolution. Non-Aqueous Reversed-Phase HPLC For very hydrophobic samples that are retained so strongly that they are not eluted with 100% acetonitrile. In this case the mobile phase is a mixture of more polar and less polar organic solvents. methylene chloride. as temperature is increased the retention of planar compounds decreases faster than non-planar compounds resulting in a change in selectivity. Selectivity properties of C-8 and C-18 Stationary Phases. C-18 etc.). When an acid or a base is ionized (converted from an uncharged to a charged molecule) it becomes much less hydrophobic and will elute much Lecture 15 3 .Figure 1. The separation of carotenoids such as carotene. Effect of Temperature In addition to the changes in selectivity brought about by changing the mobile and stationary phases. or tetrahydrofuran a commonly used as the less polar solvent (stronger solvent).

The pH at which a compound is found exactly half in the charged form and half in the uncharged form is known as the pKa. Because both groups are charged. Figure 2 shows the relative effect of mobile phase pH on the retention of a basic compound with a pKa of 4. amino acids are good examples. both the acidic and basic groups are partially charged. Some molecules. Relative Retention for a Basic Ionizable Compound at Different pH Levels. At high pH. Silanol effects can also be reduced by choosing buffer cations that are strongly held by the silanols and will not readily exchange with the analyte ions. Therefore. At low pH the molecule is fully charged (hydrophillic) and is not well retained in reversed phase systems. This property is called amphoteric. These columns are manufactured to minimize the number of very acidic silanols. At intermediate pH values. not charged Relative Retention Tim e partially charged fully charged 1 2 pKa 3 4 pH 5 6 7 Figure 2. At intermediate pH. This can result in increase retention. the molecule has its highest ionization and therefore its lowest retention at intermediate pH. It is easy to see that the pH of the mobile phase has a profound effect on the retention of ionizable compounds. the molecule is not charged (hydrophopic) and is well retained by the stationary phase. Acids lose a proton and become ionized (acquire a negative charge) as pH increases. gain a proton and acquire a positive charge as pH decreases. the molecule is found partly in the charged form and partly in the uncharged form. (all other things being equal) acids will elute earlier at higher pH and bases will elute earlier at low pH. have both acidic and basic groups on the same molecule. they can interact with the silanols of silicabased columns. Amphoteric molecules have positive charges at low pH and negative charges at high pH. When analytes are basic compounds. A molecule that is amphoteric is called a zwitterion. Therefore columns specifically designed for separating bases should be selected. Above or below this range the molecule is either charged or uncharged and its retention does not change much with changes in pH. A high pH can also be Lecture 15 4 .earlier (its k value can be reduced as much as 10-fold to 20-fold). band tailing and column-to-column variability. A low pH mobile phase can also minimize the concentration of ionized silanols. Bases on the other hand. Almost all of the pH dependent change in retention takes place 1.5 pH units on either side of the pKa value.

At high pH. silica columns may degrade. Lecture 15 5 . At higher pH. weak bases are not charged so they will not be affected by the silanols. so polymer based stationary phases should be used.used.