This the following series of our Q&A about Liquid Chromatography that was published before. This part will cover some more Q&A, which will help you to know more about liquid Chromatography.
Q: What solvent is suitable for normal phase silica gel column?
A: Both short-term and long-term storage are recommended in the mobile phase used for normal phase determination, generally n-hexane and a very small amount of isopropanol (95 / 5, 99 / 1, 99.5 / 0.5).
Q: Phosphate buffer salt depletes the column, why? Compared with other buffers, how much does the use of phosphate buffer shorten the life of the column?
A: First of all, we need to correct a misunderstanding. For chromatographic columns, pH>6.5 is not a “neutral environment” as everyone understands. When the pH of buffer salt is more than 6.5, it is an alkaline environment for silica gel column, so it is harmful to silica gel matrix. In addition, the phosphate molecule is small, which is easy to enter the bonding phase, resulting in the hydrolysis of the bonding phase. Therefore, phosphate is often used. Under the same other conditions, the service life of chromatographic column must be relatively shorter than that without phosphate buffer salt. However, phosphate also has its irreplaceable advantages, such as stable properties, strong buffer capacity and so on.
Q: How do ion pairs work in reversed phase ion pair chromatography?
A: Firstly, the dissociation end of the ion pair reagent forms an ion association complex with the target ion to reduce its polarity, so that it can be better retained on the chromatographic column. Moreover, according to the hydrophobic effect theory, the hydrophobic end of ion pair reagent is easy to interact with C18 chain, so it is easier to retain on the column. Therefore, the action of ion pair reagent is a mixed retention mechanism, that is, the ion pair retention mechanism, and a disguised reversed-phase retention mechanism formed by the formation of a close combination between the sample and the ion pair, and the ion pair reagent masking the polar groups in the compound. These two actions work together.
Q: What is the difference between tetrabutylammonium ion pair reagent and sodium alkyl sulfonate ion pair reagent? Why does tetrabutylammonium ion pair reagent have such a great influence on the service life of chromatographic column?
A: Tetrabutylammonium ion pair reagents are indeed not as widely used as sodium alkyl sulfonate plasma pair reagents. The reason is that acid polar substances can easily improve the retention capacity in reversed-phase chromatography by reducing pH value. Reducing pH can inhibit the ionization of acid, make the acid in neutral state and enhance the force with hydrophobic carbon chain. Alkali analytes are seriously affected by the upper limit of pH of silica gel matrix, and it is difficult to adjust the pH of mobile phase to very high. Therefore, cation pair reagents are often added at low pH when analyzing alkaline compounds.
Experiments show that tetrabutylammonium ion pair reagents are more difficult to remove from the packing surface than sodium alkyl sulfonate and other cation pair reagents, so the life loss of chromatographic column is serious.
Q: Why does the retention time change for the same chromatographic column, after finishing Project A and then Project B? Why is the peak shape abnormal? What will happen?
A: The chromatographic column is polluted by strong retention substances, that is, after the denaturation of the packing materials, the retention time is advanced and delayed. The specific depends on the nature of the pollutant and the joint action of the analyte, stationary phase and pollutant. The situation is more complex, and it is difficult to predict the experimental results. During normal maintenance, pay attention to backwashing and cleaning the pollutants with solvent that can dissolve them after determination, so as to reduce or avoid this situation.
Q: What is the purpose of pre column derivatization by HPLC?
A: The chemical derivatization method in chromatographic technology refers to the method of separation and detection after changing the sample components into corresponding derivatives with special chemical reagents (generally referred to as derivatization reagents or labeling reagents) in the chromatographic process. The purpose is: 1. To improve the detection sensitivity by introducing ultraviolet-visible strong absorption functional groups into the detected object or adding fluorescent groups to convert them into fluorescent derivatives; 2. Improve the separation and selectivity of analytical samples.
Q: When multiple samples are difficult to separate, how to improve the separation by selecting appropriate columns?
A: Three main influencing factors can be considered to improve the separation: column efficiency, selectivity and retention factor. The column efficiency can be improved by reducing the particle size of the packing materials and increasing the column length; The selectivity is related to the selection of stationary phase and pH conditions. The selectivity can be improved by selecting appropriate chromatographic column and appropriate pH; The retention factor is mainly related to the composition and proportion of the mobile phase. Secondly, the pH and ionic strength of the mobile phase affect the existing state of the target, and also affect the retention factor. Sometimes, properly prolonging the peak time and increasing the retention factor can also improve the seperation.
Q: When should I consider using phenyl column and cyano column?
A: Phenyl column has high selectivity for the determination of aromatic compounds containing benzene ring. CN column can be used as either a reversed-phase column with the weakest retention capacity or a normal phase column with reduced activity.
Q: The same type of column has differences in length, particle size, etc. How to choose these?
A: The length and particle size are used to change the column efficiency, and it’s ok about thes election principle if it’s enough to use. When the number of substances to be separated is ≤ 5, the chromatographic column with 150 mm column length can meet the separation of most samples.
Q: Can an ordinary C18 column be used as a normal phase chromatographic column? What kind of chromatographic column is the most commonly used and common in normal phase chromatography? What should be paid attention to in the use of normal phase column compared with reverse phase column?
A: The separation mode of normal phase chromatography means that the polarity of stationary phase is greater than that of mobile phase. Conversely, the polarity of mobile phase is reversed phase. C18 stationary phase is relatively hydrophobic. Strong hydrophobicity means that the polarity is very weak. There should be no mobile phase with weaker polarity than it. Moreover, when using the chromatographic column, it should be noted that polar stationary phase should avoid polar solvent as much as possible, and nonpolar stationary phase should avoid nonpolar solvent as much as possible, otherwise similar “similar miscibility” may effect occur.
The common columns of normal phase system include silica gel column, amino column, CN based column and diol glycol based column. The most common column should be silica gel column. The most important thing to pay attention to is the ratio of normal phase column and reversed phase column to moisture. Normal phase column is very sensitive to moisture. The slight change of moisture content in the mobile phase has a great influence on retention time. Therefore, the equilibrium time before the determination of normal phase column is several Hours or even longer.
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