Bad peak shape? Not good separation? You need to adjust the
Secret 1: from strong to weakGenerally, 90% acetonitrile (or methanol) / water (or buffer solution) is used to test, so that the separation results can be obtained quickly, and then the proportion of organic solvent (acetonitrile or methanol) can be adjusted according to the peak situation.
Secret 2: triple rule
When the amount of organic solvent (methanol or acetonitrile) is reduced by 10%, the retention factor increases by 3 times, which is a triple rule. This is a smart and labor-saving way. In the process of adjustment, pay attention to observe the separation of each peak.
Secret 3: coarse tuning and fine tuning
When the separation reaches a certain level, the 10% change of organic solvent should be adjusted to 5%, and the adjustment rate should be gradually reduced according to the rule until the separation of each component is no longer changed.
1. The properties of mobile phase require that an ideal liquid chromatography mobile phase solvent should have the characteristics of low viscosity, good compatibility with detector, easy to obtain pure product and low toxicity.
The following aspects should be considered in the selection of mobile phase
① The flow correspondingly does not change any of the properties of the packing. Ion exchange resin with low crosslinking degree and exclusion chromatography packing will swell or contract when encountering some organic phase, which will change the properties of packed bed of chromatographic column. The basic mobile phase can not be used in silica gel column system. The acidic mobile phase can not be used in the column system of alumina and magnesium oxide.
② Purity. The life of chromatographic column is related to the passing of a large amount of mobile phase, especially when impurities in solvent accumulate on the column.
③ Must match detector. When using UV detector, the mobile phase used should have no absorption or little absorption at the detection wavelength. When the differential refractive detector is used, the mobile phase should be the solvent which has great difference between the refractive index and the sample, so as to improve the sensitivity.
④ The viscosity should be low (< 2cp). High viscosity solvent will affect the diffusion and mass transfer of solute, reduce column efficiency, increase column pressure drop and prolong separation time. The best choice is the mobile phase with boiling point below 100 ℃.
⑤ The solubility of the sample should be appropriate. If the solubility is not good, the sample will precipitate on the column head, which not only affects the purification and separation, but also worsens the column.
⑥ The sample is easy to recover. Volatile solvents should be used.
2. PH value of mobile phase
When the samples of weak acid (3 ≤ pKa ≤ 7) or weak base (7 ≤ pKa ≤ 8) are separated by RP-HPLC, the dissociation of sample components is inhibited by adjusting the pH value of mobile phase, the retention of components on stationary phase is increased, and the peak shape is improved. For weak acids, the smaller the pH value of the mobile phase, the greater the K value of the component. When the pH value is far less than the pKa value of the weak acid, the weak acid mainly exists in the form of molecules; for the weak base, the situation is opposite. In the analysis of weak acid samples, a small amount of weak acid is usually added into the mobile phase, usually 50mmol / L phosphate buffer solution and 1% acetic acid solution; when analyzing weak base samples, a small amount of weak base is usually added into the mobile phase, and 50mmol / L phosphate buffer solution and 30mmol / L triethylamine solution are commonly used.
Note: the addition of organic amines in the mobile phase can weaken the strong interaction between the basic solute and the residual silanol group, and reduce or eliminate the phenomenon of peak tailing. Therefore, in this case, organic amines (such as triethylamine) are also called tailing agents or tailing agents.
(triethylamine a product in which the hydrogen atom in the ammonia molecule is replaced by three ethyls. The molecular formula is (ch3ch2) 3N. Volatile colorless liquid with an odor of ammonia. The melting point is - 114.7 ℃, the boiling point is 89.3 ℃, and the relative density is 0.7275 (20 / 4 ℃). Soluble in water, ethanol, ether and other organic solvents. Triethylamine is alkaline and can form water soluble salts with inorganic acids. It can be prepared by the reaction of N, N-diethyl acetamide and lithium aluminum hydride, and can also be synthesized by gas phase alkylation of ethanolamine. It can also be used as solvent and synthesis of quaternary ammonium compounds. )
Three How to choose the pH value of buffer solution? Before choosing the pH value of buffer, we should know the pKa of analyte first. If the pH value is higher or lower than pKa, it is helpful to get a good and sharp peak. According to HH formula: pH = PKA + log ([a -] / [a]), 99% of the compounds exist in one form when the pH value is higher or lower than PKA Good sharp peaks. It shows the transformation of its ionic form and neutral compound. PKa of benzoic acid is equal to 4.2. According to HH announcement, 99% of benzoic acid exists as neutral compound when pH value of solution is equal to 2.2, and 99% of benzoic acid exists in ionic form when pH value of buffer solution is equal to 2.2. Therefore, neutral compound is retained in reversed phase column in the form of carboxylic acid when pH value of buffer solution is equal to 2.2 General buffers and their buffer ranges. It is known from table 1 that phosphate and citrate buffers can be used for pH values equal to 2.2.
When the compound has only amino group, the selection of buffer system is very simple. Most amino compounds are protonated when the pH value is less than 9. Therefore, all solutions with pH value of 7 or lower are suitable for application. You may ask why buffer salt is still used when the pH value of water is about 7, because buffer salt helps to increase the reliability of the method, and the sharpness of chromatographic peak, and the decrease of pH value is helpful It can be seen from table 1 that any buffer solution can be applied to the analysis of amino compounds, but we think that potassium phosphate with pH value equal to 3 is most suitable for the analysis of amino compounds.
In the above two examples, potassium phosphate with pH = 3 can obtain good application. In general, it is the best buffer solution for the analysis of carboxyl and amino compounds, and we believe that potassium salt is better than sodium salt in the analysis of amino compounds.
4. Degassing of mobile phase
The mobile phase used in HPLC must be degassed in advance, otherwise bubbles will easily escape from the system and affect the operation of the pump. Bubbles also affect the separation efficiency of the column, affect the sensitivity of the detector, baseline stability, and even make it impossible to detect. (the noise increases, the baseline is unstable and jumps suddenly). In addition, oxygen dissolved in the mobile phase may react with the sample, the mobile phase and even the stationary phase (such as alkyl amine). The dissolved gas will also cause the change of the pH value of the solvent, which will bring errors to the separation or analysis results. Dissolved oxygen and some solvents (such as methanol, tetrahydrofuran) can form a complex with ultraviolet absorption. This complex can improve the background absorption (especially below 260nm), and lead to a slight decrease of detection sensitivity. But more importantly, it will cause baseline drift or ghost peak (false peak) in gradient elution. In fluorescence detection, dissolved oxygen can also cause quenching phenomenon under certain conditions, especially for aromatic hydrocarbons, fatty aldehydes, ketones and so on. In some cases, the fluorescence response can be reduced by up to 95%. In electrochemical detection (especially reduction electrochemical method), the effect of oxygen is more important.
Removal of dissolved oxygen in the mobile phase will greatly improve the performance of UV detector and improve the sensitivity in some fluorescence detection applications. The common degassing methods are: heating and boiling, vacuuming, ultrasonic, helium blowing, etc.
For mixed solvents, if the extraction or boiling method is used, the composition changes caused by the volatilization of low boiling point solvents should be considered.
Ultrasonic degassing is better than ultrasonic degassing. 10-20 minutes of ultrasonic treatment is enough for degassing of many organic solvents or organic solvent / water mixture (generally 500 ml solution needs 20-30 minutes of ultrasonic treatment), which does not affect the composition of solvent. The solvent bottle should be prevented from contacting with the bottom or wall of the ultrasonic tank, so as to prevent the glass bottle from cracking, and the liquid level in the container should not be too high above the water surface.
The off-line degassing method can not maintain the degassing state of the solvent. After you stop degassing, the gas begins to return to the solvent immediately. Within 1-4 hours, the solvent will be saturated by the ambient gas.
The on-line (in system) degassing method has no such disadvantage. The most common on-line degassing method is bubbling, in which inert gas is injected into the solvent before and during chromatographic operation. Strictly speaking, this method can't degas the solvent. It just replaces the air with an inert gas (usually helium) with low solubility. In addition, there are online degassing machines.
Generally speaking, the gas in organic solvent is easy to be removed, while the gas in aqueous solution is more stubborn. Blowing helium in solution is a very effective degassing method, which is often used in electrochemical detection. Helium is expensive, but difficult to popularize.
5. Filtration of mobile phase all solvents must be filtered by 0.45 μ m (or 0.22 μ m) before use to remove impurities, including chromatographic pure reagents (unless marked "filtered" on the label). When filtering with filter membrane, pay special attention to distinguish organic phase (fat soluble) filter membrane and water-soluble (water-soluble) filter membrane. Organic phase filter membrane is usually used to filter organic solvents. When filtering aqueous solution, the flow rate is low or the filtration cannot move. Water phase filter membrane can only be used to filter aqueous solution, and it is forbidden to use organic solvent, otherwise the membrane will be dissolved! Solvents dissolved in membrane should not be used for HPLC. For the mixed mobile phase, it can be filtered separately before mixing. If it is necessary to filter after mixing, the organic phase filter membrane is preferred. Hybrid membranes are now available for sale. 6. Storage of mobile phase the mobile phase is generally stored in glass, polytetrafluoroethylene or stainless steel container, and cannot be stored in plastic container. Many organic solvents, such as methanol and acetic acid, can leach plasticizers on the surface of plastics, resulting in solvent contamination. If the contaminated solvent is used in HPLC system, the column efficiency may be reduced. The storage container must be tightly covered to prevent the change of composition caused by solvent volatilization, and prevent oxygen and carbon dioxide from dissolving into the mobile phase.
Phosphate and acetate buffer solution is easy to mold, so it should be prepared fresh as far as possible, and should not be stored. If it is necessary to store it, it can be refrigerated in the refrigerator and used within 3 days. It should be filtered again before use. The containers should be cleaned regularly, especially the bottles containing water, buffer and mixed solution, to remove the sediment of impurities at the bottom and the microorganisms that may grow. Since methanol has antiseptic effect, the bottle containing methanol has no such phenomenon.
6. Special attention should be paid to halogenated organic solvents. Halogenated solvents may contain trace acidic impurities and can react with stainless steel in HPLC system. The mixture of halogenated solvent and water is easy to decompose and can not be stored for a long time. Halogenated solvents (such as CCl4, CHCl3, etc.) mixed with various ethers (such as ether, diisopropyl ether, tetrahydrofuran, etc.) may react to produce some products which are more corrosive to stainless steel. This mixed flow should not be used or freshly prepared. In addition, when halogenated solvents (such as CH2Cl2) are mixed with some reactive organic solvents (such as acetonitrile), crystallization will occur. In short, halogenated solvents are best prepared and used fresh. If it is mixed with dry saturated alkanes, there will be no similar problem.
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