What is the difference between positive phase, reverse phase and super high efficiency in high performance liquid chromatography

High performance liquid chromatography can be divided into 3 categories according to the pressure level: normal-phase high performance liquid chromatography (pressure 5000-6000psi), reversed-phase high performance liquid chromatography (pressure 9000-10000psi) and ultra-high performance liquid chromatography (ultra-high performance liquid chromatography) (pressure 12000, even up to 15000psi).

1, regular phase high performance liquid chromatography This is not a commonly used high performance liquid chromatography method
.

The normal-phase high performance liquid phase uses a non-polar solvent as the mobile phase and silica gel particles as the stationary phase
.

In conventional high performance liquid chromatography, polar compounds adhere to polar silicones in the stationary phase for longer than non-polar compounds
.

As a result, non-polar compounds elute faster
in normal high performance liquid chromatography.

The pore size is generally around
3 microns.

Principle of normal phase chromatography technology: the use of polar stationary phase (such as polyethylene glycol, amino and nitrile bonding phase); The mobile phase is a relatively non-polar hydrophobic solvent (alkanes such as n-hexane, cyclohexane), and ethanol, isopropanol, tetrahydrofuran, tri-* methane, etc.
are often added to adjust the retention time
of the components.

It is commonly used to separate compounds with medium polarity and strong polarity (such as phenols, amines, carbonyl groups and amino acids).

2.
Reversed phase high performance liquid chromatography This is a common type of
high performance liquid chromatography.

In the reversed-phase high performance liquid phase, the stationary phase is modified by hydrocarbon chains (typically 8-18 carbon long), causing the column to become non-polar
.

Polar solvents
are also used.

In reversed-phase high performance liquid chromatography, polar molecules in the sample create a strong attraction to the solvent, allowing them to elute
more quickly.

They also do not interact with non-polar stationary phases
.

Non-polar compounds interact with stationary phases to produce van der Waals forces and dispersions, so they elute more slowly
.

Principle of reversed-phase chromatography technology: generally use non-polar stationary phase (such as C18, C8); The mobile phase is water or buffer, and methanol, acetonitrile, isopropanol, acetone, tetrahydrofuran and other organic solvents that are mutually soluble with water are often added to adjust the retention time
.

Suitable for the separation of non-polar and less polar compounds
.

RPC is more widely used in modern liquid chromatography, accounting for about
80% of the entire HPLC application.

3, ultra-high performance liquid chromatography such as high performance liquid chromatography runs faster, uses fewer solvents and has higher
resolution.

Columns are also smaller, with much
smaller size of stationary phase particles.

Smaller pores (sub-2 microns) promote better molecular separation and resolution
.

Shorter columns also allow for rapid inspection.


However, ultra-high performance liquid chromatography cannot be used for dried or unfiltered samples because smaller wells can clog, which is an expensive and time-consuming repair process
.

New advances in filler materials have begun to create intermediate solutions
between high performance liquid chromatography and ultra high performance liquid chromatography.

Core-shell particles are one of these fillers currently on the market
.

Using coreshell particles is as efficient as using sub-2 micron particles, but can operate at much lower pressures
.

There is even speculation that these efficiencies can be generated
on standard high performance liquid chromatography machines.

HPLC machines typically include a UV detector to measure the UV emitted
by molecules in a sample.

However, some HPLC machines can be used
in conjunction with mass spectrometers (MS).

When using HPLC-MS, molecules can be identified by the m/z ratio without knowing the retention time
.