Micron Spherical Silica Gel Powder for Cosmetics Additive and Column Chromatography
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Review of Practical Application of Silica Gel in Column Chromatography(II)

3. Column selection


Commonly used columns can be divided into: pressurized, normal pressure, and reduced pressure.


Pressure can increase the flow rate of the solvent and reduce the time for product collection, but it will also reduce the number of plates in the column. Therefore, when

other conditions are the same, atmospheric pressure columns are highly efficient, but they also take a long time. For example, the separation of natural compounds can

take several months for one column.


① The pressure reduction column can reduce the usage of silica gel, and it feels like it can save half or even more. However, due to the large amount of air passing through

the silica gel, the solvent will evaporate (sometimes water vapor will condense outside the column).

And some things that are easier to decompose may not be available, and a water pump must be used at the same time (which makes a lot of noise and takes a long time).


② Pressurized column is a better method, similar to normal pressure column, except that external pressure makes the solvent move faster. The pressure can be provided

by compressed air, a double-connected ball or a small air pump (the one that supplies air to the fish tank will do). It is especially suitable for separation of easily

decomposed samples. The pressure should not be too high, otherwise the solvent will move too fast and the separation effect will be reduced. Pressurized columns are

more suitable for the separation of common organic compounds.


③Normal pressure column chromatography is a common type of chromatography. Its outstanding advantages are that the separation efficiency is much higher than the

classic chemical separation method. Compared with other chromatographic methods, it does not require expensive instruments and equipment, it is convenient to replace

the mobile phase and adsorbent, consumes less materials, and has low cost. It is suitable for separating various samples with a wide range of sampling quantities from

grams to micrograms, so it is still widely used in chemical laboratories.


4.Solvent selection


The selection of solvent may be the most difficult part in the entire column chromatography separation operation, and it is also the key to the success of the experiment.

The solvent is usually screened using a simple silica gel thin layer plate.


4.1 Thin layer plate spotting


Dissolve the sample in a low-boiling organic solvent (such as chloroform, acetone, methanol, ethanol, etc.) and then use a capillary or micro-spotting tube to spot the

test solution onto the thin layer plate. The amount of spotting should be appropriate, generally, if the amount of sample spotted is smaller, the separation will be clearer,

but attention should be paid to the detection sensitivity.


4.2 Spotting and unfolding


4.2.1 Selection of developing agent


When selecting a developing agent, micro-ring method and mini-chromatography can be used to select.


① Micro-circle method: Spot the substance to be separated on the thin plate according to the conventional method, with a distance of 2-3cm between the two points,

and then use a capillary tube to draw the experimental solvent system and place it vertically in the center of the sample spot,

let the solvent flow out from the capillary in a neutral direction for development, and develop color after drying. Observe the separation of spots. When the test

substance is an unknown compound, it is often developed with a low-polarity solvent first. If the pattern remains in the dots, you need to increase the polarity of the

solvent or increase the amount of eluent. If the movement is too fast, you must use a lower polarity solvent to adjust.


②Small-scale chromatography. Use an ordinary carrier chip to make a thin plate. Place the substances to be separated on the thin plate, place it in a small glass jar or

wide-mouth bottle, unfold it, develop color after drying, and observe the spot separation.


4.2.2 Expansion method


The development methods of thin layer chromatography include upward method, downward method, two-way development method, radial development method, etc.:


① Ascending method: Make the spreading agent climb up from bottom.


②Downward method: Make the developing agent from top to bottom. This method can use a less polar developing agent.


③ Two-way expansion method: Take a square thin plate and conduct two-way expansion like paper chromatography.


④ Radial expansion method: The adsorbent can be painted into a circle or fan shape, and a developing agent can be added to the center of the circle to expand the thin

layer radially.


⑤Multiple development: Use the developing agent to develop the thin layer once. This is called single development. After one development, take out the thin plate,

evaporate the developing agent and then develop again.


⑥Gradient development: The composition of the developing agent used in this method is continuously changing. Generally, a buret containing a highly polar developing

agent can be inserted deep into a sealed chromatography tank containing a weakly polar developing agent,

use an electromagnetic stirrer to mix the dropped highly polar developing agent in the tank. At this time, the polarity of the developing agent gradually changes from

weak to strong, so that the mixture of multiple components with large polarity differences can be well separated.


4.3 Expand color development


After unfolding, if the substances are colored, their positions can be clearly observed on the thin layer. If they are colorless, certain methods must be used to develop

the color and position it, while determining the location and size of the spots.


4.4 Polarity of commonly used solvents


The order of polarity of commonly used solvents is:

petroleum ether < cyclohexane/hexane < benzene < diethyl ether < chloroform < ethyl acetate < n-butanol < acetone < ethanol < methanol < water.


Dielectric constant is generally used to describe polarity: petroleum ether (none) (cyclic) hexane (1.88) benzene (2.3) ether


(4.5) Chloroform (5.2) Ethyl acetate (6.1) Acetone (21.5) <Ethanol (25.8) <Methanol (31.2) <Water (81.0).


In thin layer chromatography, when the polarity of the solvent is too high, all the substances to be separated will be close to the solvent front. When the polarity of the

solvent is too small, almost all the substances to be separated will remain in the dots. In both cases, the material to be separated cannot be separated.


Using a single solvent often cannot achieve good separation results. Mixed solvents are often used. A mixed solvent composed of high polarity and low-grade solvents is

often used. Highly polar solvents also have the effect of increasing discrimination.