Preparation of Liquid Chromatography Column Two types of 10b cone preparative liquid chromatography columns were prepared: column 1 [50mm@(18→9)mm.id.] And column 2 [100mm @ (36 → 18) mm.id .]. The column material of the visualization device is plexiglass. Since plexiglass is easily corroded by solvents, stainless steel chromatographic columns with the same processing size are used to investigate the column efficiency. When investigating the flow pattern, in order to observe a clear sample band, the injection volume of column 1 was 80LL, and the injection volume of column 2 was 600LL. When separating the samples, the injection volumes were 40 and 160LL, respectively.
Flow pattern research Because the entire visualization device is an integrated structure, the position of the inlet and outlet of the internal cone-shaped liquid chromatography column can be accurately determined. Paste the paper ruler vertically on one of the vertical planes. The 0 scale position is the same as the position at the entrance of the cone-shaped liquid chromatography column. With this ruler, you can accurately measure the distance the sample band moves along the column length.
The dynamic process of the band in the column can clearly see the stereoscopic image of the band flow pattern with the naked eye. However, due to the limitation of the digital camera recording system, only two-dimensional photos can be given. It is a dynamic photo of the band flow pattern obtained by the visualization device. It can be seen that the sample can maintain a flat plug flow pattern during the elution in the cone column. In (A), amorphous silica packing is used, and the sample has obvious tailing in the column, which broadens the band and reduces the efficiency of the separation column. (B) Spherical silica gel packing is used, and the sample has no tailing. The measurement results of the pore size distribution of the amorphous filler show that it contains a large number of pore distributions around 3nm, which causes tailing due to the strong retention effect on small molecular samples, and this pore distribution in spherical silica monomers is rare.
As can be seen from the intuitive band flow photo, (B) has a higher column efficiency than (A). The peak shape of (A) is severely tailed, and the peak shape of (B) is very symmetrical. This result is consistent with the dynamic picture of the band flow type directly captured by the visualization device, indicating that the visualization device can truly reflect the sample spectrum Dynamic process carried in the column.
The effect of flow velocity on the band flow pattern The flow velocity of the mobile phase may affect the band flow pattern, that is, the flat plug flow pattern obtained by the cone column. The experiment used a digital camera and a self-made ruler to investigate the effect of flow velocity on the flow pattern. The results are shown below. The results show that, for a cone-shaped column with a column length of 5 cm and a cone angle of 10 b, the change in flow rate has almost no effect on the band flow pattern. stable. Comparing curves a and b, it can be seen that the maximum length of the spherical silica filler can keep the band as a plug is longer than the length of the amorphous filler, indicating that the spherical filler is better than the amorphous filler.
The effect of the particle size of the filler on the flow pattern was to investigate the effect of the particle size of the filler on the plug-shaped flow pattern in the 10b cone column, and the separation process of the sample on the 20Lm spherical silica gel filler was observed. It is a dynamic photo of the band flow pattern (flow rate 2mL / min). It can be seen that the flat plug shape can be maintained throughout the chromatographic process, indicating that the particle size of the filler has basically no effect on the flat plug shape flow pattern in the cone column. However, in the case of the same injection volume and flow rate, the width of the band in the column of the small particle packing is small. It is the corresponding UV detection spectrum when the particle size of the filler is different. (A) has a half-width of 0126min, and (B) has a half-width of 0119min. This is consistent with the intuitively taken band-flow dynamic photos, which shows The visualization device can provide an intuitive and effective method for evaluating the column efficiency of the prepared liquid chromatography column.
The effect of column size on the flow pattern is to investigate the effect of the column size on the flow pattern. The above-mentioned conical column with a good plug-like flow pattern (cone angle remains unchanged) is scaled up to obtain a conical column with a column length of 10 cm. It can be seen that in the enlarged cone-shaped column, the sample remains as a flat plug during the entire separation process, and the change of the flow rate will not affect the flow pattern of the band. It can be seen from the photo of the band flow pattern that the sample is evenly distributed at the entrance of the column, indicating that the design of the column head and the distribution plate is reasonable.
The results show that when the 10b cone liquid chromatography column is scaled up in equal proportion, the total column efficiency is increased in proportion, and because the effect of the extra-column effect is reduced, the column efficiency is also improved.
In order to compare the effect of column size change on resolution, the same chromatographic packing and mobile phase (mobile phase 3) were used to separate carmine and brilliant blue on column 1 and column 2, respectively. In order to obtain the ultraviolet detection spectrum, it can be seen that the resolution Rs1 of the two samples on column 1 is 1152, the resolution Rs2 on column 2 is 2121, and Rs2 / Rs1 = 1145, which is slightly higher than 2 (11414). It shows that the size of the 10b cone-shaped column is slightly enlarged after scaling up.
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