Determination of 3-amino-4-hydroxybenzaldehyde content

I. Requirements for the properties of the mobile phase in liquid chromatography: An ideal solvent for the mobile phase in liquid chromatography should have low viscosity, good compatibility with the detector, ease of purification, and low toxicity. After selecting the packing material (fixed phase), a strong solvent reduces the adsorption of the solute on the surface of the packing material, resulting in a decrease in the capacity factor k ； Weaker solvents increase the adsorption of the solute on the surface of the filler, resulting in an increased capacity factor k. Therefore, the k value is a function of the mobile phase composition. The number of plates N is generally inversely proportional to the viscosity of the mobile phase. Therefore, the following aspects should be considered when selecting the mobile phase: ① The mobile phase should not change any properties of the packing. Ion exchange resins with low cross-linking density and size-exclusion chromatography packing materials may swell or shrink in the presence of certain organic phases, thereby altering the properties of the chromatography column packing. Alkaline mobile phases cannot be used in silica gel column systems. Acidic mobile phases cannot be used in column systems with adsorbents such as alumina and magnesium oxide. ②Purity. The lifetime of the chromatography column is related to the large amount of mobile phase that passes through it, especially when impurities present in the solvent accumulate on the column. ③It must match the detector. When using a UV detector, the mobile phase used should have no absorption, or only very low absorption, at the detection wavelength. When using a differential refractometry detector, a solvent with a refractive index significantly different from that of the sample should be chosen as the mobile phase to improve sensitivity. ④The viscosity should be low (the mobile phase used in normal-phase chromatography typically consists of alkanes with an appropriate amount of polar modifier added. The mobile phase in reverse-phase chromatography is usually based on water, to which a certain amount of polar modifiers that are miscible with water is added, such as methanol, acetonitrile, **furan, etc. The properties of the polarity modifier and its proportion have a significant impact on the retention value and separation selectivity of the solute. Under normal circumstances, the methanol-water system can meet the separation requirements for most samples; moreover, its low viscosity and low cost make it the most commonly used mobile phase in reverse-phase chromatography. However, Snyder recommends using an acetonitrile-water system for initial experiments, as compared to methanol, acetonitrile has a higher solvent strength and lower viscosity, and it can meet the requirements for detection at wavelengths of 185–205 nm in the ultraviolet range. Therefore, overall, the acetonitrile-water system is superior to the methanol-water system. When separating multi-component samples with large polarity differences, gradient elution is also required to ensure that each component has an appropriate k value and is separated well. In reverse-phase chromatography, if the same set of samples is to be separated within the same time frame, the ratio of the flushing strength when using methanol/water as the flushing agent relates to the flushing strength ratios of acetonitrile/water or **furan/water as follows: C_acetonitrile = 0.32C_methanol + 0.57C_methanol; C**furan = 0.66C_methanol. C represents the volume percentage of the various organic solvents mixed with water. The rinsing strength of 100% methanol is equivalent to that of 89% acetonitrile/water or 66% **furan/water. IV. Filtration of the mobile phase in liquid chromatography: All solvents must be filtered through 0.45 μm (or 0.22 μm) filters before use to remove impurity particles, and this applies equally to chromatographically pure reagents (unless it is indicated on the label that they are \"already filtered\"). When using filter membranes, it is particularly important to distinguish between organic-phase (liposoluble) filter membranes and aqueous-phase (water-soluble) filter membranes. Organic phase filter membranes are generally used to filter organic solvents; when filtering aqueous solutions, the flow rate is low or filtration is not possible. Aqueous-phase filter membranes can only be used to filter aqueous solutions; they must not be used with organic solvents, otherwise the filter membrane will dissolve! Solvents containing filter membranes must not be used in HPLC. For mixed mobile phases, they can be filtered separately before mixing; if filtration after mixing is required, an organic-phase filter membrane is the preferred choice. Mixed-type filter membranes are now available for sale. V. Degassing of the liquid chromatography mobile phase: The mobile phase used must be degassed in advance; otherwise, bubbles may form within the system, affecting the operation of the pump. Bubbles can also affect the separation efficiency of the column, as well as the sensitivity of the detector and the stability of the baseline; in some cases, they can even prevent detection. (Noise increases, baseline is unstable, sudden fluctuations). Furthermore, oxygen dissolved in the mobile phase may also react with the sample, the mobile phase, and even the stationary phase (such as alkyl amines). Dissolved gases can also cause changes in the pH of the solvent, leading to errors in separation or analysis results. Dissolved oxygen can form complexes with certain solvents (such as methanol and **furan**) that exhibit ultraviolet absorption. These complexes increase the background absorption (especially below 260 nm), leading to a slight decrease in detection sensitivity. Importantly, they can cause baseline drift or the formation of spurious peaks during gradient elution. In fluorescence detection, dissolved oxygen can also cause quenching under certain conditions, especially for aromatic hydrocarbons, aliphatic aldehydes, and the like. In some cases, the fluorescence response can be reduced by up to 95%. In electrochemical detection (especially reduction electrochemistry), the influence of oxygen is greater. Removing dissolved oxygen from the mobile phase will **improve the performance of the UV detector and also enhance sensitivity in some fluorescence detection applications. Common degassing methods include: heating and boiling, vacuum pumping, ultrasound, helium blowing, etc. For mixed solvents, if evaporation or boiling is used, the composition changes caused by the volatilization of the low-boiling-point solvent must be taken into account. Ultrasonic degassing is a good method; 10–20 minutes of ultrasonic treatment is sufficient for degassing many organic solvents or mixtures of organic solvents and water (typically, 500 ml of solution requires 20–30 minutes of ultrasonic treatment), and this method does not affect the composition of the solvent. During ultrasonication, care should be taken to avoid contact between the solvent bottle and the bottom or walls of the ultrasonic tank, so as to prevent the glass bottle from breaking; moreover, the liquid level in the container should not be too high above the water surface. The offline (outside the system) degassing method cannot maintain the degassed state of the solvent; once degassing is stopped, gases immediately begin to return into the solvent. Within 1 to 4 hours, the solvent will be saturated with ambient gases again. The online (in-system) degassing method has no such drawback. The most commonly used online degassing method is bubbling, which involves injecting an inert gas into the solvent before and during chromatographic operation. Strictly speaking, this method cannot degas the solvent; it merely replaces the air with an inert gas of low solubility (usually helium). There are also online degasers. Generally, gases in organic solvents are easy to remove, while gases in aqueous solutions are more persistent. Blowing helium into the solution is a quite effective degassing method, and this continuous degassing approach is often used in electrochemical testing. But helium is expensive and difficult to make widespread use of. VI. Storage of liquid chromatography mobile phase The mobile phase is generally stored in glass, polytetrafluoroethylene, or stainless steel containers; it should not be stored in plastic containers. Many organic solvents such as methanol and acetic acid can leach out the plasticizers on the surface of plastics, resulting in solvent contamination. If such contaminated solvents are used in HPLC systems, it may lead to a decrease in column efficiency. The storage container must be tightly sealed to prevent changes in composition due to solvent evaporation, as well as to prevent oxygen and carbon dioxide from dissolving into the mobile phase. Phosphate and acetate buffers tend to mold easily; they should be prepared fresh whenever possible and not stored. If storage is indeed necessary, it can be kept refrigerated in the fridge and used within 3 days; it should be filtered again before use. Containers should be cleaned regularly, especially bottles that hold water, buffers, and mixed solutions, in order to remove impurity deposits at the bottom as well as any microorganisms that may have grown. Since methanol has preservative properties, bottles containing methanol do not exhibit this phenomenon. Please read the content carefully, and select an appropriate mobile phase based on the properties of the substance you need to analyze. If that doesn’t work, you can turn to the liquid chromatography column supplier for assistance

Method: Reverse-phase high-performance liquid chromatography was used, with a Nucleosil 100—5C18 column as the stationary phase. The mobile phase consisted of phosphate buffer (pH 3.0) – acetonitrile – **furan (85:10:5), at a flow rate of 0.9 mL·min^-1, and the detection wavelength was 254 nm. Acetylate 3-ASA in the sample using derivatization