Conventional Column Chromatography
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3.Chromatographic Column (Manual)
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Description
Technical Parameters
Conventional column chromatography utilizes the difference in distribution ratios of different substances between the stationary phase and the mobile phase, or the different retention capacities of fixed phases relative to different substances, to achieve substance separation. When the mixture flows through the chromatographic column with the mobile phase, it will interact with the stationary phase in the column (such as dissolution, adsorption, etc.). Due to the differences in physical and chemical properties and structures of each component in the mixture, the magnitude and strength of their interactions with the stationary phase also vary. Under the same driving force, the retention time of each component in the stationary phase is different, so that the components in the mixture flow out of the column in a certain order, achieving separation.
Parameter
Pesticide residue detection
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We often say that Conventional column chromatography is actually column chromatography separation, also known as column chromatography. It was first invented and used by Russian scientists in 1903. It crushed a solution of plant pigments into calcium carbonate powder and found different color bands on the calcium carbonate powder from top to bottom. By separating and extracting different color bands, relatively pure chlorophyll, lutein, etc. were obtained.
After a hundred years of development, this method has become relatively mature, and the stationary phase has also evolved from a single calcium carbonate powder to alumina (which can be divided into acidic, neutral, alkaline), silica gel, activated carbon, etc. Among them, silica gel is the most commonly used in major laboratories, generally divided into normal phase silica gel and reverse phase silica gel!
The stationary phase (silica gel) generally needs to be purified and activated, and the particle size should be uniform. In theory, the smaller the particle size and the larger the surface area per unit mass, the higher the adsorption capacity and the better the separation effect.
However, in practical use, when the fixed phase particles are too small, there are often two situations: one is that they are lighter in weight and easily float in the air (silica gel cannot be degraded by the human body, and the finer, the more toxic it is); Secondly, small particles can also lead to small gaps between particles, resulting in a slow solvent flow rate and time consumption; Taking silicone as an example, the largest commercially used size of silicone is currently 200-300 mesh.
In terms of column passing methods, in addition to the automatic analysis equipment such as forward circulation preparation, efficient liquid phase preparation, and column passing machines mentioned earlier, manual column passing is mainly divided into three types: atmospheric pressure column passing, depressurization column passing, and pressurization column passing.
Among them, the depressurization through the column results in poor adsorption efficiency due to the fast flow rate of the eluent, which leads to the loss of most of the number of trays and poor separation efficiency. It is suitable for separating samples with relatively open TLC plates, especially for samples with only one large polar impurity in the product; Most of its time is spent on the pre-treatment of compounds, removing some impurities before careful purification. It should be noted that solvent evaporation caused by negative pressure during use can lead to the condensation of a large number of water droplets on the outer surface of the column. Water sensitive compounds should not be easily attempted.
Atmospheric pressure column passing is the most effective separation method among the three, but because the solvent often moves too slowly, even the best separation effect can affect efficiency. Therefore, pressure is usually applied at this time, which is called pressurized column passing; But the pressure should not be too high. Generally, a double chain ball is used for manual operation, and a fish pump is used for automatic operation. Both can be modified as shown in the picture.
Conventional column crossing process:
Preparation work should be done well:
When selecting a column, observe whether there is a sanding board. For columns without sanding boards, remember to fill the bottom with some cotton. The cotton should not be too thick or too thin, as long as it can block the silicone from coming off. (The blogger directly dismantled a clothes hanger and used it to insert cotton into the outlet on the plug)
Prepare the minimum polarity eluent, UV lamp (recommended for laboratory personnel, handheld, ready to take a photo at any time), pressure equipment, and test tube rack as necessary!
Select a suitable column, add silicone gel, soak the silicone gel, and flatten it
The selection of conventional column chromatography is very important, and their size should be calculated based on the amount of product, which can generally be divided into:
A few milligrams of column (the final choice for total synthesis);
A column of several tens of milligrams (methodological expansion substrate);
500 milligrams to 1 gram of column (using methodology as raw material);
Pillars around 5G and larger scales.
The most common ratio of column diameter to height in the laboratory is generally between 1:2.5 and 1:15. The amount of silica gel used as a stationary phase during sample loading is about 20 to 40 times that of the sample. The standard for selecting columns should be based on the TLC plate climbing situation of your sample. If there are many impurities in close proximity, try to choose a larger column as much as possible, so that your sample can be covered with a small thin layer (recommended thickness below 5mm). If the impurities are far away, this thickness can naturally be increased, but it is not recommended that the thickness of your sample exceed 2cm, unless the product is simply washed.
Many people make a mistake of thinking that adding more silicone, even if it is thicker on my sample, can it be separated well? This answer is negative!
The reason is that these people never thought that the samples below and above were not running on the same line, so they could only obtain some pure ones, most of which were crossed, not to mention impure ones, wasting time and solvents!
That's also why the sample requires thinning!
After selecting the column, adding silicone can be done through a feeding funnel. At the same time, be sure to wear a mask and operate in a fume hood. The reason for this will not be elaborated by the blogger (silicosis).
As for the infiltration of silica gel, the recommended process is: first, place the silica gel column with the silica gel installed vertically, flatten the surface of the silica gel with your hand, connect the bottom of the silica gel column to a water pump, pump out the silica gel, and then pour in the least polar eluent from above. When the eluent is about to leak out, close the shut-off valve to prevent solvent from being pumped into the water pump.
Then, under pressure, the silicone gel can be uniformly soaked by flushing seven or eight column volumes with the smallest amount of developing agent.
Sample above
Sampling is divided into wet sampling and dry sampling.
(1) Wet sampling.
The recommended method is to use a pipette with a rubber tip to suck the sample, close to the top section of the silicone gel, slowly and evenly drip the sample until it covers a layer, then stop adding the sample and apply some pressure to immerse the oily substance into the silicone gel layer; Immerse the oily substance into the silicone layer and repeat this process multiple times until all samples are loaded!
Some new students may be curious why this operation is carried out instead of coating the wet process oil like substance all at once?
In fact, this operation can reduce the effective height of the sample layer. At this point, column chromatography has already begun, and the subsequent samples are like solvents pushing the previous samples to adsorb and desorb in silica gel.
(2) As for dry sampling.
Unless absolutely necessary, it is not recommended. Solid samples are more recommended to be beaten or crystallized. "Unscrupulous methods, ethyl acetate vs. water, ultrasonic oscillation assisted crystallization of high boiling oil" and "Sharing a laboratory recrystallization method" are recommended.
Dry sampling generally brings two problems; On the one hand, during the sample mixing process, the possibility of reaction between the sample and silica gel increases due to the need to raise the temperature for rotary evaporation; On the other hand, dry loading means that adsorbents will be introduced into the sample layer, increasing the height of the sample layer or the size of the column, requiring more time and more solvent.
If there is no other choice, simply pour the adsorbed powder slowly into the column for loading, with some requirements similar to wet methods.
Add anhydrous sodium sulfate or quartz sand
In order to avoid impacting the sample layer during the addition of eluent, causing unevenness, deformation, and affecting separation efficiency, a layer of anhydrous sodium sulfate or quartz sand is generally laid on top of the sample layer.
Our engineers personally prefer to lay anhydrous sodium sulfate because it also has a drying function and is friendly to solvents such as dichloromethane, which are prone to volatilization, heat absorption, and condensation!
Gradient elution, sampling and collection
After loading, first rinse the column with a small polar solvent (a solvent that will not allow the product to pass through) for 2-3 column volumes (one column volume is defined as the volume of the mobile phase flowing in and out) to ensure uniformity, and then select a proportion for elution.
How to choose the eluent that passes through the column?
The ratio of eluent is the ratio when your product reaches an Rf value of about 0.2 on the TLC plate
This is the experience of column washing that I have read many books and shared, and the actual process is also very similar. Personally, I prefer to reduce the gradient by twice.
For example, if the solvent for TLC plate climbing is PE/EA=2:1, the blogger is more likely to start gradient elution from PE/EA=4:1 and end at PE/EA=2:1, generally resulting in better results.
There are also techniques when collecting eluent. You can first choose a larger test tube to receive it, and when TLC is approaching the target point, switch to a smaller test tube. This can reduce the possibility of receiving more and avoid impurities.
If you encounter situations where impurity points and product points are close together, especially those with weak fluorescence, remember not to apply pressure. Instead, choose to pass the column under normal pressure, and change the tube every time half or less tubes are connected, which can also increase the purification probability.
Remember not to handle the pillar in a timely manner!
Many people have a habit of assuming that the column has been passed and dried when there is no product in one or two tubes after TLC in the later stage.
I only realized that the yield was too low when I calculated it, or when I went to make a spectrum, I found that the spectrum was incorrect and missed the wrong point. I regretted it deeply.
So it is recommended that you do not rush to process the column after completing the desired point. Instead, perform nuclear magnetic resonance to match the yield before processing.
If you are busy or in a hurry to purify other compounds with this conventional column chromatography, it is recommended to rinse with a high polarity benign mixed solvent and leave this part of the eluent separately for identification results.
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