Adsorption chromatographyIt is the one that is used moreChromatographyMethod is a commonly used separation method in chemical experiments.Especially suitable for many mediummolecular weightSeparation of samples (low volatile samples with molecular weight less than 1000), especiallyLiposolubilityComponents are generally not applicable to high molecular weight samples such asprotein、PolysaccharideOr ionic hydrophilicitychemical compoundSeparation of etc.The separation effect of adsorption chromatography depends onAdsorbent、solventAnd the nature of the separated compound.
fcp silica gelIt is a porous substance withsiloxaneAt the same time, there are many silanol groups on the surface of the particles.The adsorption of silica gel is related to the content of silanol group.Silanol group can absorb water through the formation of hydrogen bond, so the adsorption capacity of silica gel decreases with the increase of absorbed water.If the water absorption exceeds 17%, the adsorption capacity is extremely weak and can not be used as an adsorbent, but can be used as a supporting agent in distribution chromatography.For the activation of silica gel, when the silica gel is heated to 100~110 ℃, the water adsorbed on the surface of silica gel due to hydrogen bonding can be removed.When the temperature rises to 500 ℃, the silanol group on the surface of silica gel can also be dehydrated and condensed into siloxane bond, thus losing the activity of water adsorption due to hydrogen bond, so it no longer has the nature of adsorbent, and its adsorption activity can not be recovered even though it is treated with water.Therefore, the activation of silica gel should not be carried out at a higher temperature (generally above 170 ℃, a small amount of bound water is lost).
Silica gel is an acidic adsorbent, suitable for chromatography of neutral or acidic components.At the same time, silica gel is a weak acidCation exchangerThe silanol group on its surface can release weakly acidic hydrogen ions. When encountering strong alkaline compoundsIon exchange reactionAnd adsorbed alkaline compounds.
alumina
aluminaIt may be alkaline (because it can be mixed with sodium carbonate and other ingredients), which is ideal for the separation of some alkaline Chinese herbal ingredients, such as alkaloids.However, alkaline alumina is not suitable for separation of aldehydes, ketones, vinegar, lactones and other types of compounds.Because sometimes alkaline aluminum oxide can have secondary reactions with the above components, such as isomerization, oxidation, elimination reaction, etc.The bright alkaline impurities in alumina can be removed by washing with water to neutral, which is called neutral alumina.Neutral alumina still belongs to the category of alkaline adsorbent, which is applicable to the separation of acidic components.Treating aluminum oxide with dilute nitric acid or dilute hydrochloric acid can not only neutralize the alkaline impurities contained in aluminum oxide, but also make the surface of aluminum oxide particles bear the anions of NO3 - or CI -, so it has the property of separation from the exchanger and is suitable for chromatography of acidic components. This aluminum oxide is calledAcid alumina。Alumina for chromatography is used for column chromatography, and its particle size is required to be between 100 and 160 meshes.The particle size is 100 meshes, and the separation effect is poor: less than 160 meshes, the flow rate of solution concentration is large and slow, and the spectral band is easy to diffuse.The dosage ratio of the sample to alumina is generally between 1:20 and 50. The ratio of the inner diameter of the chromatographic column to the length of the column is between 1:10-20.
When washing the column with solvent, the flow rate should not be too fast. The flow rate of eluent is generally appropriate when the number of milliliters of liquid flowing out every half to one hour is equal to the weight (g) of adsorbent used.
Activated carbon
Activated carbonIt is a kind of non-polar adsorbent that is widely used.Generally, it needs to be washed first with dilute hydrochloric acid, then with ethanol, and then with water. It can be used for chromatography after drying at 80 ℃.The activated carbon used for chromatography should preferably be granular activated carbon injection. If it is fine activated carbon powder, a proper amount of diatomite should be added as a filter aid and loaded into the column to avoid too slow flow rate.Activated carbon is mainly used to separate water-soluble components, such as amino acids, sugars and some glycosides.Activated carbon has adsorption effect, which is strongest in aqueous solution, but weaker in organic solvent.Therefore, the elution ability of water is the weakest, while that of organic solvents is stronger.For example, when eluting with alcohol water, the elution force increases with the increase of ethanol concentration.Activated carbon pairAromatic compoundThe adsorption force of is greater thanAliphatic compoundsThe adsorption capacity of macromolecular compounds is greater than that of small molecular compounds.These differences in adsorption can be used to separate water-soluble aromatic substances from aliphatic substances, monosaccharides from polysaccharides, and amino acids from peptides.
solvent
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During chromatographysolventThe choice of is very important for component separation.The solvent (single agent or mixed solvent) used in column chromatography is conventionally calledEluentIt is often called developing agent for thin layer or paper chromatography.The selection of eluent must be considered according to the combination of the separated substance and the nature of the selected adsorbent. When polar adsorbent is used for chromatography, when the separated substance is a weak polar substance, the weak polar solvent is generally used as eluent;If the separated substance is a strong polar component, polar solvent must be selected as eluent.If a weak adsorbent is used for a polar substance (such as diatomite or talcum powder instead of silica gel), the polarity of the eluent must also be reduced accordingly.
During column layer operation, the method can be used for adding the separated sample, or a suitable solvent can be selected to dissolve the sample and add it.The solvent used to dissolve the sample should be of low polarity so that the separated components can be adsorbed.Then gradually increase the polarity of the solvent.This polarity increase is a very slow process, called "gradient elution", so that the components adsorbed on the chromatographic column are eluted one by one.If the polarity increases too much (the gradient is too large), satisfactory separation cannot be obtained.The elution ability of solvent, sometimes the dielectric constant of solvent can be used（ε）To represent.The higher the dielectric constant, the greater the elution ability.The above elution sequence is only applicable to polar adsorbents, such as silica gel and alumina.For non-polar adsorbents, such as activated carbon, the adsorption in water or hydrophilic solvent is stronger than that in fat soluble solvent.
Separation property
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The separated substance, adsorbent and eluent together constitute the three elements of adsorption chromatography, which are closely connected with each other.Under the condition of specified adsorbent and eluent, the separation of each component is directly related to the structure and property of the separated material.For polar adsorbent, the polarity of components is large and the adsorption is strong.
