Mr. Huang! thanks for ur reply but methanol, acetonitrile n tetrahydrofuran used as mobile phases in reversed mode are also organic in nature
Dr. Khan- advantages for what?
There's a lot of prep work done on silica, but with automated (and non-automated) flash chromatography systems. The columns are inexpensive, and are generally considered disposable.
Many sample are soluble in polar organic solvents and water, so people use C18 because of a common conception that silica dissolves in thise solvents (methanol and water). Note that Waters (and others) do sell a line of HILIC (aqueous normal phase) columns that are bare silica.
I wouldn't say that reverse-phase is better than normal phase, it just works most of the time so people have the columns readily available. People tend to use what works most of the time.
As usually happens with equipment and devices, advantages and disadvantages (pros & cons) are upon your needs/goals. Perhaps what you need is a comparison table.
If reversed mode is not advantageous over normal phase then why it was developed? and why more than 80% methods are developed in reversed mode?
It was developed as an alternative method to normal phase chromatography to provide different selectivity. For the most part, compounds are retained sufficiently well for the experiment; those that are stuck on the column can be generally washed out with dichloromethane.
Why are 80% of methods developed? it simply works sufficiently well that prople use it. For the other 20%, other methods are needed.
Many compounds are purified with flash chromatography, where ~80% of the purifications are done on silica.; the purity is often checked on an LC-MS running reverse phase showing that many compounds could be run either way. The big advantage of reverse phase is that it simply works sufficiently well for the vast majority of the compounds. People aren't going to find the perfect chromatography, just something that works for their needs. We are generally too busy to go much beyond "works sufficiently well"
There are several reasons why reverse phase HPLC has become more of a standard means of HPLC separation than normal phase. Reverse phase columns have a hydrophobic stationary phase which works well for retention of most organic analytes. This also means that water can be used as a mobile phase in conjuntion with less polar solvents such as MeCN and MeOH which can be adjusted at highly controlled rates to improve chromatographic performance. Gradient separations in normal phase are much more complicated because of UV cutoff variation as well as differences in compressability of common hydrophobic solvents, which would have an effect on flow rate. Reverse phase chromatography also has the advantage of being able to use pH selectivity to improve separations. There are also many more choices in stationary phases for reverse phase vs. normal phase.
So the short answer is revese phase offers many more options to a chromatographer, but it depends on your analyte(s), detection method and what type of separation you are trying to achieve.
in addition, for MS coupling, RP is much more easier to use.
however, nowadays, with current technologies RP and NP can be used for analysis and purification. However, for highly apolar compounds NP is usually much better.
I compared both rapidly, and it seems that overload limits is much higher with RP compared to NP. And silica columns are less stable than C18 columns.
And I feel that classical NP HPLC column are less efficient that RP HPLC column. However they are good alternatives.
Historically, I think that natural purification were used with a first silica gel fractionnation with further purification using an orthogonal strategy such as RP HPLC.
Unfortunately there is not much informations about all that...
RP (HPLC) column is highly stable and efficient. Thus, these advantages explain the most frequent use of RP in HPLC application in these days. RP could be applied to separation of a very wide range of molecules including charged and polar molecules. It also allows precise control of variables such as organic solvent type and concentration, and pH.
NP is more useful when your analyte is more polar and when we talking about the RP no doubt it is time saving and more efficient but the important thing is that what sort of separation you looking for then you can decide the phase...
In RP HPLC, you are able to save money. It allows you to use water as a solvent together with other solvents.
RP is NOT "preferred" over NP chromatography. Such a statement makes no sense (no context is provided) as the mode of chromatography used is determined mostly by the properties of the sample. Some modes of chromatography do have aspects that make them very attractive for specific goals. For example, if you are running a large scale preparative HPLC analysis and wish to collect large volume fractions, then removal of the mobile phase is important. Buffers would make this very time consuming as would solvents with high boiling points like water. If the sample is soluble in a highly volatile NP solvent then this will be much easier to evaporate off and collect the sample material.
Water IS the "Universal Solvent". Analysis requires that samples be fully soluble or dissolved in the mobile phase for analysis. This is why RP is so common (because most samples can be initially dissolved in water or a high percentage of aqueous solution).
- Badges
- Science method