Selection&Optimization Strategies of Liquid Phase Mobile Phase

***information collect from internet

一.Selection&optimization strategies of liquid phase mobile phase

Principle 1: From strong to weak

Generally, the test is carried out with 90% acetonitrile (or methanol)/water (or buffer solution) first, so that the separation results can be obtained quickly, and then adjust the proportion of organic solvent (acetonitrile or methanol) according to the peaks.

Principle 2: The Rule of Threes

For every 10% reduction in the amount of organic solvent (methanol or acetonitrile), the retention factor is increased by a factor of approximately 3. This is known as the Rule of Threes. This is a smart and labor-saving approach. During the adjustment, pay attention to the separation of the individual peaks.

Principle 3: Coarse adjustment to fine adjustment

When the separation reaches a certain degree, the 10% change of organic solvent should be adjusted to 5%, and according to this rule gradually reduce the adjustment rate until the separation of the components no longer change.

一.The nature of the mobile phase requirements

An ideal liquid chromatography mobile phase solvent should have low viscosity, good compatibility with the detector, easy to get pure products and low toxicity and other characteristics.

The following aspects should be considered when selecting the mobile phase:

①The mobile phase should not change any properties of the packing material.

Ion exchange resins and exclusion chromatography packings with low cross-linking degrees sometimes dissolve or shrink when encountering certain organic phases, thus changing the nature of the column packing bed. Basic 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.

② Purity

The lifetime of a chromatographic column is related to the passage of a large amount of mobile phase, especially when impurities contained in the solvent accumulate on the column.

③It must be matched to the detector

When using a UV detector, the mobile phase used should have no absorption at the detection wavelength, or very little absorption. When using an oscillometric refractive detector, a solvent with a large difference between the refractive coefficient and the sample should be selected as the mobile phase to improve sensitivity.

④ The viscosity should be low (should be <2cp)

High viscosity solvents will affect the diffusion and mass transfer of solutes and reduce the column efficiency, and will also increase the column pressure drop and prolong the separation time. It is best to choose a mobile phase with a boiling point below 100°C.

⑤ The solubility of the sample should be appropriate

If the solubility is not good, the sample will precipitate at the head of the column, which not only affects the purification and separation, but also deteriorates the column.

⑥.The sample is easily recovered

?Volatile solvents should be used.

二.The pH of the mobile phase

When separating weak acid (3≤pKa≤7) or weak base (7≤pKa≤8) samples by reversed-phase chromatography, the technique of adjusting the pH of the mobile phase in order to inhibit the dissociation of the sample components, to increase the retention of the components in the stationary phase and to improve the peak shape is known as reversed-phase ion suppression technique.

For weak acids, the smaller the pH value of the mobile phase, the larger the k value of the component, when the pH value is much smaller than the pKa value of the weak acid, the weak acid exists mainly in the form of molecules; for weak bases, the situation is reversed. When analyzing weak acid samples, a small amount of weak acid is usually added to the mobile phase, commonly used 50mmol/L phosphate buffer and 1% acetic acid solution; when analyzing weak base samples, a small amount of weak base is usually added to the mobile phase, commonly used 50mmol/L phosphate buffer and 30mmol/L triethylamine solution.

Note: The addition of organic amines to the mobile phase attenuates the strong interaction of the basic solute with the residual silanol groups and reduces or eliminates peak tailing. Therefore, organic amines (e.g. triethylamine) are also called tailing agents or tail removers in this context.

(Triethylamine triethylamine The product of substitution of hydrogen atoms in the ammonia molecule by 3 ethyl groups. Molecular formula (CH3CH2)3N. volatile colorless liquid with ammonia odor. Melting point -114.7 ℃, boiling point 89.3 ℃, relative density 0.7275 (20 / 4 ℃).

Soluble in water and organic solvents such as ethanol and ether. Triethylamine is alkaline and can produce water-soluble salts with inorganic acids. It can be produced by the reaction of N,N- diethylacetamide and lithium aluminum hydride, and can also be synthesized by gas-phase alkylation reaction with ethanolamine. It can be used as rubber vulcanization accelerator, wetting agent and fungicide, etc. It can also be used as solvent and for the synthesis of quaternary ammonium compounds.

三.How to choose buffer pH

Before selecting the buffer PH value, you should first understand the analyte Pka, higher or lower than Pka two units of PH value, helps to obtain a good, sharp peaks, from the HH formula: PH = Pka + log ([A-]/[A]) know that the solution PH is higher or lower than the Pka two units of compounds in 99% of the compound in the form of a form of compounds to be present in order to obtain good sharp peaks. Shown is the shift between its ionic form and the neutral compound, the Pka of benzoic acid is equal to 4.2, and theoretically it is known by the HH metric that 99% of the benzoic acid exists as a neutral compound when the solution pH is equal to 2.2, and 99% of the benzoic acid exists as an ionic form when the pH is equal to 6.2, so that when the buffer pH is equal to 2.2, the neutral compound is retained as a carboxylic acid on the reversed-phase column.

When the compound is only amine, the choice of buffer system is very simple, most amine compounds are protonated at pH values less than 9, so all solutions with a pH value of 7 or less are suitable for the application, you may ask why buffer salts are used when the pH value of water is about 7, because buffer salts help to increase the reliability of the method, as well as the sharpness of the peaks, and the lowering of the pH value helps to weaken the retention of the amines and reduces the retention of compounds. The decrease in pH helps to minimize the retention of amino compounds and reduces the interaction of the compounds with the silica hydroxyl groups on the silica gel surface, resulting in sharper peaks.

四.Degassing of mobile phase

The mobile phase used for HPLC must be degassed beforehand, otherwise air bubbles will easily escape from the system, affecting the work of the pump. Air bubbles will also affect the separation efficiency of the column, affect the sensitivity of the detector, baseline stability, or even make it impossible to detect. (Increased noise, baseline instability, sudden jumps). In addition, oxygen dissolved in the mobile phase may react with the sample, the mobile phase or even the stationary phase (e.g. alkylamines). Dissolved gases can also cause changes in the pH of the solvent, bringing errors to the separation or analysis results.

Dissolved oxygen can form UV-absorbing complexes with certain solvents (e.g., methanol, tetrahydrofuran), which can increase the background absorption (especially below 260 nm) and lead to a slight decrease in detection sensitivity but, more importantly, can cause baseline drift or the formation of ghost peaks (pseudo-peaks) during gradient elution. In fluorescence detection, dissolved oxygen can also cause quenching under certain conditions, especially for aromatic hydrocarbons, aliphatic aldehydes, and ketones. In some cases, the fluorescence response can be reduced by up to 95%. In electrochemical detection (especially reductive electrochemical methods), the effect of oxygen is even greater.

Removing dissolved oxygen from the mobile phase will greatly improve the performance of the UV detector and will also improve sensitivity in some fluorescence detection applications. Commonly used degassing methods include heating and boiling, evacuation, sonication, and helium blowing.

For mixed solvents, changes in composition due to volatilization of low-boiling solvents need to be considered if pumping or boiling is used.

Ultrasonic degassing is better, 10 ~ 20 minutes of ultrasonic treatment for many organic solvents or organic solvents / water mixture of degassing is sufficient (generally 500ml solution requires ultrasound 20 ~ 30min before), this method does not affect the composition of the solvent. Ultrasonication should be careful to avoid solvent bottles and ultrasonic tank bottom or wall contact, so as to avoid the glass bottle rupture, the liquid level in the container should not be too high above the water surface.

The off-line (out-of-system) degassing method does not maintain the degassed state of the solvent, and the gas begins to return to the solvent immediately after you stop degassing. Within 1 to 4 hours, the solvent will again be saturated with ambient gas.

On-line (in-system) degassing does not have this disadvantage. The most commonly used on-line degassing method is bubbling, in which an inert gas is sprayed into the solvent before and while the chromatography operation is in progress. Strictly speaking, this method does not degas the solvent; it simply replaces the air with a low solubility inert gas (usually helium). There are also in-line degassers.

Generally speaking, gases in organic solvents are easy to degas, while gases in aqueous solutions are more recalcitrant. Blowing helium into the solution is a fairly effective degassing method, and this continuous degassing method is often used in electrochemical testing. However, helium is expensive and difficult to popularize.

五.Filtration of mobile phase

All solvents must be filtered through a 0.45μm (or 0.22μm) membrane to remove impurity particles prior to use, including chromatographically pure reagents (unless labeled as “filtered”). When using membranes for filtration, it is important to distinguish between organic phase (fat soluble) membranes and aqueous phase (water soluble) membranes. Organic phase membranes are generally used for filtering organic solvents, while aqueous membranes do not flow at a low rate or do not filter. Aqueous phase membranes can only be used for filtration of aqueous solutions, and are strictly prohibited for use with organic solvents, otherwise the membrane will be dissolved! Solvents dissolved in the membrane should not be used in HPLC. For mixed mobile phases, they can be filtered separately before mixing, but if they need to be filtered after mixing, organic phase membranes are preferred. Mixed membranes are now available.

六.Storage of mobile phase

The mobile phase is generally stored in glass, PTFE or stainless steel containers and cannot be stored in plastic containers. Many organic solvents such as methanol and acetic acid can leach plasticizers from plastic surfaces, resulting in contamination of the solvent. This contaminated solvent, if used in an HPLC system, may cause a decrease in column efficiency. Storage containers must be tightly covered to prevent volatilization of solvents causing compositional changes, and to prevent oxygen and carbon dioxide from dissolving into the mobile phase.

Phosphate and acetate buffers are susceptible to mold growth and should be prepared as fresh as possible and not stored. If storage is necessary, it can be frozen in the refrigerator and used within 3 days, and should be re-filtered before use. Containers should be cleaned regularly, especially bottles containing water, buffer and mixed solutions, to remove impurity deposits at the bottom and possible growth of microorganisms. This is not the case with bottles containing methanol because of its preservative effect.

七.Special attention to halogenated organic solvents

Halogenated solvents may contain traces of acidic impurities that can react with stainless steel in HPLC systems. Mixtures of halogenated solvents with water decompose relatively easily and should not be stored for too long. Halogenated solvents (such as CCl4, CHCl3, etc.) and a variety of ethers (such as ether, diisopropyl ether, tetrahydrofuran, etc.) mixed, may react to generate some of the stainless steel has a greater corrosive product, this mixed flow should be as far as possible not to use, or freshly prepared. In addition, halogenated solvents (such as CH2Cl2) and some reactive organic solvents (such as acetonitrile) mixed with static, but also produce crystallization. In short, halogenated solvents are best used freshly formulated. Similar problems do not arise if they are mixed with dry saturated alkanes.