In High Performance Liquid Chromatography, why are ratios of solvents used?

Question

How do they allow for better separation? For example, why isn't 100% hexane used?

Answer 1

Not sure if I can fulfill Ed's "real answer". I like the word you used for tuning solvent polarity- a knob. Modern students may understand this better.

There is no theoretical restriction in chromatography to use multiple solvents or a single solvent. For example in gas chromatography, you always use a pure gas. The reason for using a mixture of solvents in liquid chromatography originates from the concept of "general elution problem of chromatography" which basically says that there is no single column or a single mobile phase which can separate everything. Not only separation scientists use a single mixture composition of mobile phase (isocratic), they can also continuously change the mobile phase composition as a function of time. This is called gradient elution.

When you do real separations, you have no idea beforehand about the solubility of analytes injected into the column. If these injected solutes are not soluble in a pure solvent they will permanently absorb on the column. A real sample may contain 30-40 compounds. Say a petroleum sample, literally have thousands and thousands of different molecules. Now think yourself: will they all dissolve in 100% hexane?

Answer 2

Mixture of solvents is used for 2 main reasons:

Elution time

For given HPLC column and set of analytes, the mobile phase must have the proper degree of general polarity, what is often called "solvent strength".

For polar sorbents like silica or alumina, more polar solvents have generally bigger strength with shorter elution times. Note that the solvent strength is compound specific.

For non polar sorbents with silica substituted by C8 or C18 alkyl chains, common for RP ( reversed phase ) HPLC, the strength order is reversed as well.

The ideal elution time is 2-4 times the column "dead time" of passing the column without any retention.

If compounds leave the column too fast they would not be separated well.

If they leave the column too slowly, elution would take too long time and chromatographic peaks would be too broad.

In the case of RP HPLC, pure methanol may lead to too short elution times, pure water to too long times.

Selectivity

Various analytes have various interaction with particular solvents, leading to various elution times.

Major contributions to solvent polarity are:

• dipole dipole interaction
• solvent acidity ( Bronsted, Lewis )
• solvent basicity ( Bronsted, Lewis )

That leads to different positions of chromatographic peaks and their mutual separation for various mobile phases.

Each pure solvent has its own combination of the above contributions. The optimal combination usually happens to be somewhere between properties of pure solvents, therefore mixing is necessery.

For the common reversed phase HPLC with non polar column, the frequent procedure to determine the proper mobile phase is this.

1. Find the methanol-water solution to get elution time of the main/reference component as 2-4 times the dead time.
2. If peak separation is not satisfactory, find the solutions acetonitrile-water and tetrahydrofurane-water with the about same elution time of the main/reference component. The rule of thumb is: 50% methanol, 40% acetonitrile and 30% THF have about the same general "solvent strength".
3. Try combinations of the above 3 mobile phases to get the optimal separation of peaks.
4. In case the analytes have significant acido-basic behaviour, there are usually used buffer solutions, instead of pure water, for better and consistent distinguishing of their dissociation.
5. There is often used gradient HPLC with progressively increasing solvent strength to compress chromatograms. But it is usable for shifting the solvent selectivity as well.

See Using a Solvent Triangle to Optimize an HPLC Separation

Selectivity in reversed phase LC separations - part 1 - solvent-type selectivity.

Answer 3

It depends on what you want to separate or purify on your column as well as what type of column you have.

Generally, you will run many normal phase columns with varying mixtures of hexane and ethyl acetate and most reverse phase columns with acetonitrile and water or methanol (each lab usually has its two standard solvents for given HPLC systems and rarely deviates). Each solvent has a certain polarity as does the column. Neither of these are variable. But the compound you want to purify can have all sorts of polarities from the polar end of what is separable on said column to the nonpolar end.

For most compounds, there will be a ratio of the two solvents generally used that is ‘best’ for separation, whichever goal is most important (time saving, purity, a certain persistent impurity to be removed or something else). But it does happen now and again that the ideal solvent for a given application is ‘100 % A’ or ‘100 % B’. I have had it happen to me but I can’t recall what the system was.

Outside of the isocratic argument above, many HPLC runs are gradient runs, meaning you start at 100 % A and end at 100 % B (or whichever ratios are best for your application). This allows for a better overall separation, especially when you have both polar and less polar compounds to separate. Of course, running gradients is impossible with only one solvent.