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    Home > Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Their Applications

    Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Their Applications

    • Last Update: 2018-08-27
    • Source: Internet
    • Author: User
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    According to the difference of relative polarity between mobile phase and stationary phase, liquid chromatography can be divided into positive phase and reverse phase When the polarity of mobile phase is greater than that of stationary phase, it is called reversed phase chromatography Because of its high column efficiency, strong separation ability and clear retention mechanism, RP-HPLC has become the most widely used separation mode in liquid chromatography, which is suitable for the separation and purification of various polar or non-polar compounds, such as alkaloids, sugars, fatty acids, steroids, nucleic acids, amino acids, polypeptides, proteins, etc In reversed phase chromatography, the commonly used stationary phase is the bonding of various functional groups on silica gel matrix, including C18, C8, C4, phenyl, cyano, amino, etc C18 is the most commonly used functional group in these bonded phases According to statistics, more than 80% of reversed-phase chromatographic separation and purification uses C18 bonded phase Therefore, C18 chromatographic column has become the most commonly used and necessary general-purpose chromatographic column in each laboratory Although C18 column has a very wide range of application, however, for some samples with high polarity or strong hydrophilicity, common C18 column may encounter some problems in the separation and purification of such samples In reversed-phase chromatography, the commonly used eluting solvents can be sorted according to their polarity as follows: water < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol In order to obtain a better retention time for such samples (strong polarity or strong hydrophilicity) on the separation column, it is usually necessary to use a high proportion of water phase system as the mobile phase When using pure water system (including pure water or pure salt solution) as mobile phase, the long carbon chains of C18 column will choose to avoid boiling water and mix with each other, resulting in the instantaneous retention capacity of chromatographic column decreased or even no retention effect This phenomenon is called hydrophobic collapse (see the left part of Figure 1) Although this situation is reversible (it can be washed with methanol or acetonitrile and other organic solvents), it will cause certain damage to the chromatographic column, so it is necessary to avoid this situation as much as possible Fig 1 The surface bonding phase diagram of common C18 column and c18aq column silica gel In view of the above situation, the manufacturer of chromatographic packing has made technical improvement By modifying the surface of silica gel matrix, such as introducing hydrophilic cyano (see the right part of Fig 1), the hydrophilicity of silica gel surface is stronger, so as to prevent the occurrence of hydrophobic collapse The modified C18 column is named as c18aq column, i.e hydrophilic C18 column, which is specially designed for high water phase elution conditions and can withstand 100% water phase system C18aq column has been widely used in the separation and purification of organic acids, peptides, nucleosides and water-soluble vitamins A typical application of c18aq column in flash preparation of purified samples is desalting operation, that is, to remove the salt or buffer components in the sample solvent, so as to facilitate the sample application in subsequent research In this case, the highly polar brilliant blue was separated and purified on the c18aq column as a sample, so that the solvent of the sample was replaced from the buffer solution to the organic solvent, thus facilitating the subsequent spin evaporation and other operations, saving the solvent and operation time Some impurities in the sample were removed, and the purity of the sample was improved Figure 2 Chemical structure formula of the sample The sample used in this paper is brilliant blue FCF The purity of the crude product is 86% The molecular structure formula is shown in Figure 2 Take 300mg of powdered brilliant blue crude solid, dissolve it in 1m NaH 2PO 4 aqueous solution, shake well, and make the sample completely dissolved into clear and transparent solution Use syringe to sample the sample solution to flash separation column, and the desalination and purification parameters of flash separation column are shown in Table 1 Table 1 Sepabean ® machine 2 chromatographic column 12 g sepaflash ® C18 reversed phase separation column (spherical silica gel, 20-45 μ m, 100 μ m, order No.: sw-5222-012-sp) 12 g sepaflash ® c18aq reversed phase separation column (spherical silica gel, 20-45 μ m, 100 μ m, Order No.: sw-5222-012-sp (AQ)) detection wavelength 254 nm mobile phase solvent A: water; solvent B: methanol flow rate 30 ml / min injection volume 300 mg (86% purity of bright blue crude product) elution gradient time (CV) solvent B (%) time (CV) solvent B (%) 0 10 Results and discussion Firstly, we use c18aq column to desalinate and purify the sample We set a step gradient to maintain the pure water system as the mobile phase and run 10 column volumes (CVS) Observing the chromatogram (as shown in Figure 3), we can see that when pure water is used as the mobile phase, the sample is completely retained on the c18aq column Next, methanol in the mobile phase was directly increased to 100%, and 7.5 column volumes were maintained at this gradient The sample was eluted at about 11.5 to 13.5 column volumes In the collected components, the sample solution has been replaced with methanol from the NaH 2PO 4 buffer solution Compared with the high proportion of aqueous solution, it is obvious that methanol is easier to be removed in the subsequent spinning treatment, so as to obtain the target product for the next study Figure 3 Flash purification chromatogram of sample on c18aq column in order to compare the retention behavior of c18aq column and common C18 column for strong polar sample, we conducted parallel contrast test Since the water phase ratio of common C18 column is about 90%, the initial gradient is set as 10% methanol / 90% water The flash purification chromatogram of the sample on the common C18 column is shown in Figure 4 According to figure 4, due to the hydrophobic collapse of stationary phase caused by high proportion of water phase, the sample is basically not retained on the common C18 column, and is directly eluted by the mobile phase, so the desalination or separation and purification operation cannot be completed Figure 4 Flash purification chromatogram of sample on common C18 column compared with linear gradient, step gradient can bring the following benefits: 1 Save solvent and running time for separation and purification; 2 The elution peak of the target product presents a narrow peak, which reduces the volume of the collected components, so as to facilitate the subsequent rotary evaporation treatment and save time; 3 The collected target product dissolves in methanol, and the solvent is easy to be evaporated, thus saving the drying treatment time of the product Therefore, for the separation and purification of samples with strong polarity or hydrophilicity, sepaflash ® c18aq reversed-phase separation column combined with sepabean ® machine rapid liquid chromatography system provides a fast and efficient solution for the rapid separation and purification of such samples Sepaflash ® c18aq reversed phase separation column series products launched by Santai technology have a variety of specifications (see Table 2) Table 2 Sepaflash ® c18aq reversed phase separation column parameters (packing: high efficient physical C18 (AQ), 20 – 45 μ m, 100 Å ) Item Number Column Size Flow Rate (mL/min) Max.Pressure (psi/bar) SW-5222-004-SP(AQ) 5.4 g 5-15 400/27.5 SW-5222-012-SP(AQ) 20 g 10-25 400/27.5 SW-5222-025-SP(AQ) 33 g 10-25 400/27.5 SW-5222-040-SP(AQ) 48 g 15-30 400 / 27.5 sw-5222-080-sp (AQ) 105 g 25-50 350 / 24.0 sw-5222-120-sp (AQ) 155 g 30-60 300 / 20.7 sw-5222-220-sp (AQ) 300 g 40-80 300 / 20.7 sw-5222-330-sp (AQ) 420 g 40-80 250 / 17.2 to learn more about sepabean ® machine Please visit CBG online store for detailed specifications or ordering information of flash purification column  
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