Archive for the ‘Kromatografi’ Category

Many HPLC methods can be used with an LC-MS with little or no modification. However, there are some things that don’t work, and some things where you may find that slight changes improve your signal wonderfully:

  • LC-MS relies on drying away all the solvent. It therefore follows that you cannot use solvents with non-volatile components. Phosphate buffers and salts are bad. They will crystalize out as the solvent is dried away, and fill the ion source with solid gunk. Ion exchange chromatography is therefore unlikely to work. But you can try changing non-volatile salts to volatile ones, such as ammonium acetate.
  • All the solvent must be dried away, so the less solvent there is, the better. Consider reducing your flow rate. Electrospray mass spec signals depend on concentration, not total amount of stuff squirted into source per minute, so reducing flow rate will also increase sensitivity!
  • You are no longer dependant on perfect separation of peaks, and good retention time, for the identification of your chemical of interest. But the machines are more expensive and in demand. Consider sacrificing a little separation for improved throughput. Most LC-MS people use very short columns, 100mm maximum, often less.


What matters to retention time is how fast the solvent flows linearly along the column. You can calculate a flow rate for a narrower column very easily.
The cross-sectional area of the column is πr2
The cross-section of a 4.6 mm column is about five times that of a 2.1 mm column.
Therefore you should use a flow of about one fifth, i.e. 200 µl.min-1 instead of 1000µl.min-1

Note that 200 µl.min-1 is absolutely ideal for most mass specs. Buy 2mm columns instead of 4.6mm where ever possible!


Written by : Dr. Lionel Hill (JIC, UK)

In electrospray ionisation the metabolites are sprayed out of a fine needle, positioned with its end poking into a “cage” formed by metal surround.

There is a big voltage between the needle and the cage, so the little sprayed droplets find themselves in a strong electric field.

There are various theories about how it works. This is the oldest (and best): The ions already floating around in the droplets repel each other, just like the hairs on a child’s head when a physics teacher attaches him/her to the Van der Graff generator.

They find themselves on the outside of the droplet. But meanwhile a stream of drying gas, often heated, is blown around the spray area. The droplets shrink as the solvent evaporates, and the charged ions are forced closer and closer together.

Eventually the repulsive forces between the ions overcome the surface tension of the droplet, and it “explodes” into many tiny droplets, whose solvent disappears even more quickly.

Soon the ions are left free in an atmosphere of drying gas. The important requirement is that the ions should be present in solution before the electrospray process begins. This method requires a high flow of drying gas. In contrast to APCI (Atmospheric Pressure Chemical Ionisation), the drying gas does all the work of removing solvent. The pressure at the nebulising needle needn’t be big, because the needle sticks right into the cage close to where the ions are released, and doesn’t have far to squirt.

The ions that are seen tend to be the ions that this metabolite would normally form in solution. Fortunately, the electrospray needle does act like the electrode in a battery and allow some electrochemistry to go on, creating higher concentrations of these ions than you might normally expect. This is good, as it means you can still detect carboxylic acids by their -COO group, even in the presence of some formic acid, which would normally force most of the groups into their -COOH uncharged state.

Incidentally, as in APCI, the need to form a fine spray does influence the choice of solvent. Some solvents that aren’t usually used in LC-MS are rejected because they don’t have the surface tension characteristics for ES (Electrospray) to work properly.

Atmospheric Pressure Chemical Ionisation

In APCI the sample is sprayed out of a fine needle that is usually a bit shorter than that used in Electrospray. The solvent is then dried away by a heater. The remaining solutes, hopefully also vaporised, are then blown towards a corona discharge, which I think is just a technical name for a spark.

In the spark the vaporised solvent and analyte are ionised. The solvent can play an important role here, because if it has a lower affinity for protons than the analyte, it can pass a proton onto an analyte molecule, creating an [M+H]+ ion.

Since the metabolite of interest has to float its way into the spark in a stream of gas, this method relies on some volatility (unlike electrospray). Ionization events are comparatively rare, so the chances of an ionized metabolite meeting another ionizing species and getting a second charge are even rarer: most ions in APCI are singly charged.

Written by : Dr. Lionel Hill (JIC, UK)


An LC-MS is an HPLC system with a mass spec detector. The HPLC separates chemicals by conventional chromatography on a column. Usually the method will be reverse phase chromatography, where the metabolite binds to the column by hydrophobic interactions in the presence of a hydrophilic solvent (for instance water) and is eluted off by a more hydrophobic solvent (methanol or acetonitrile). As the metabolites appear from the end of the column they enter the mass detector, where the solvent is removed and the metabolites are ionised. The metabolites must be ionised because the detector can only work with ions, not neutral molecules. And ions only fly through a very good vacuum, so removal of the solvent is a vital first step. The mass detector then scans the molecules it sees by mass and produces a full high-resolution spectrum, separating all ions that have different masses.

Why chromatography?

Since mass spectrometers can differentiate a lot of chemicals all mixed together on the basis of mass, it is tempting not to bother separating chemicals first with hplc. There are however two good reasons why chromatography is a very good thing.
  • Most biologically interesting chemicals exist as isomers. Isomers have exactly the same mass and cannot normally be differentiated by a mass detector, no matter how expensive it is. Therefore it helps if you can additionally separate the isomers before hand by chromatography.
  • When a mixture of chemicals enters the process of ionisation, the chemicals can interact and affect one another’s chances of getting properly ionised. This is called ion suppression. It is usually a problem where you are trying to detect one minor, or poorly ionised chemical in the presence of a large amount of something else, maybe a buffer from the sample. Some pre-purification of the ionisation mixture can get the suppressed away from the suppressors. There are ways to recognise ion suppression.

And why is the chromatography usually reverse-phase?

The first thing that happens to the flow from the HPLC when it enters the mass detector is that the solvent is squirted through a fine needle and evaporated away, to leave metabolites floating free. If the solvent contains non-volatile salts, they will appear, solid and horrible and clog up the system. Therefore ion-exchange chromatography is not really compatible with LC-MS. If you must have salts present, low concentrations of volatile things, such as ammonium acetate, can be tolerated.

Why bother with mass spectroscopy?

HPLC separates things, but provides little extra information about what a chemical might be. In fact, it is hard in hplc to be certain that a particular peak is pure, and contains only a single chemical. Adding a mass spec to this tells you the masses of all the chemicals present in the peak, which can be a very good starting point for identifying them, and an excellent method to check for purity. You can also use even a simple mass spec as a mass-specific detector, specific for your chemical of interest.

More sophisticated mass detectors such as triple quadrupole and ion-trap instruments can be set up to carry out more detailed structure-dependant analyses on what is eluting from the HPLC system.

After HPLC separation the sample goes straight into a mass detector. Mass specs detect ions in a vacuum, so the first tasks in the LC-MS are to

  • remove the solvent and ionize the metabolites.
  • get the ions into a vacuum.

Historical note:
GC-MS came before LC-MS because it is comparatively easy to pump off a small amount of GC carrier gas, but quite difficult to pump off all the vapour that can be created from even a small amount of liquid. One ml water will produce 1.3 litres of vapour at room temperature and pressure…


Written by : Dr. Lionel Hill, John Innes Centre UK