Why is HPLC going faster and faster, and why is the peak time of HPLC peaks getting earlier and more

Can you give us a little more hints?

The peak time is early, whether it is reflected in the middle of the day, or a period of time, a period of time.

The most likely thing that happens is that the pump for your highly polar mobile phase is a little clogged, causing the elution to get stronger and stronger, and that's the temperature difference over the course of the day, and the system isn't in good balance.

There are several factors that affect the timing of the peak: the mobile phase ratio, the pH of the mobile phase buffer, the flow rate, the column temperature, and the particle size of the column.

In your case: (1) The first few stitches of the instrument are not well balanced. (2) The internal damage of the column collapses (3) See if the pressure of the instrument panel is stable and whether the pipeline is blocked? If the sample solution is too dirty, it is also easy to block the assembly line.

Because of the high separation efficiency, the separation efficiency is not only the number of theoretical plates, but also the length of time to separate the sample, because it is higher than the number of theoretical plates of ordinary liquid chromatography, ion chromatography, gas chromatography, etc., and the separation speed is fast, so it is called high-performance liquid phase, and ultra-high performance liquid phase is that the separation efficiency is higher than that of high-performance liquid phase, which is mainly determined by the smaller particle diameter of the column packing and the higher pressure of the separation system.

HPLC is High Performance Liquid Chromatography, and the full name in English is High Performance Liquid Chromatography. This method is used as an important separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection and forensic inspection.

Uses: High performance liquid chromatography is more suitable for the separation and analysis of substances with high boiling point, poor thermal stability, physiological activity and relatively large relative molecular weight, so it is widely used in the analysis of nucleic acids, peptides, lactones, fused cyclic aromatic hydrocarbons, polymers, drugs, human metabolites, surfactants, antioxidants, pesticides, herbicides and other substances.

Principle: High performance liquid chromatography uses liquid as the mobile phase, adopts a high-pressure infusion system, pumps a single solvent with different polarities or mixed solvents, buffers and other mobile phases with different polarities into the chromatographic column equipped with stationary phase, and after the components in the column are separated, they enter the detector for detection, so as to realize the analysis and separation of the sample.

Operation method: As shown in the figure below, the mobile phase in the solvent receptacle is sucked in by the pump, mixed and then output by the gradient controller according to a certain gradient, the pressure and flow rate are measured, and the injection valve (device) is introduced into the detector after the protection column and the separation column are tested, the data is processed by the data processing equipment or the chromatogram is recorded by the recorder, the fraction collector collects the fraction, and the waste bottle collects the waste liquid.

High performance liquid chromatography (HPLC) is also known as "high performance liquid chromatography", "high performance liquid chromatography", "high resolution liquid chromatography", "modern column chromatography", etc.

High performance liquid chromatography is an important branch of chromatography, using liquid as the mobile phase, using a high-pressure infusion system, a single solvent with different polarity or different proportions of mixed solvents, buffers and other mobile phases are pumped into the chromatographic column equipped with stationary phase, and after the components in the column are separated, they enter the detector for detection, so as to realize the analysis of the sample.

Principle and operation method:

The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector, and is loaded into the chromatographic column (stationary phase) by the mobile phase.

1. Concept:

HPLC refers to high performance liquid chromatography, and the full name in English is High Performance Liquid Chromatography. It is also known as "high-pressure liquid chromatography", "high-speed liquid chromatography", "high-resolution liquid chromatography", "modern column chromatography", etc.

High performance liquid chromatography is an important branch of chromatography, using liquid as the mobile phase, using a high-pressure infusion system, a single solvent with different polarity or different proportions of mixed solvents, buffers and other mobile phases are pumped into the chromatographic column equipped with stationary phase, and after the components in the column are separated, they enter the detector for detection, so as to realize the analysis of the sample.

This method has become an important application of separation and analysis technology in the fields of chemistry, medicine, industry, agronomy, commodity inspection and forensic inspection.

2. Principle: Liquid chromatography can be divided into: liquid-solid adsorption chromatography, liquid-liquid partition chromatography, ion exchange chromatography, ion-pair chromatography, molecular exclusion chromatography or gel permeation chromatography according to the different separation mechanisms.

3. Application of HPLC.

In terms of environmental protection, HPLC can be applied to:

1) Pesticide residue analysis: chlorine-containing pesticides, organophosphorus pesticides, pyrethrum, etc.;

2) Carcinogens: nitrofuran and its metabolites, mycin, nitrosamines, benzopyrene, etc.;

3) Water quality analysis: endocrine pollutants, etc.;

In biochemistry, HPLC can be applied to:

1) Protein analysis;

2) analysis of peptides;

3) nucleic acids; 4) amino acids ;

High performance liquid chromatography (HPLC) is an efficient and rapid analytical separation technology developed rapidly in the seventies of the last century, and is an important means of modern separation testing.

The separation principle of chromatography is that when each component dissolved in the mobile phase passes through the stationary phase, due to the different size and strength of the interaction (adsorption, distribution, exclusion, affinity) with the stationary phase, the residence time in the stationary phase is different, and then it flows out of the stationary phase successively. It is also known as chromatography and chromatography.

Operation method: 1. Filter the mobile phase and select different filter membranes according to your needs.

2. Ultrasonic degassing of the filtered mobile phase for 10-20 minutes.

3. Open the HPLC workstation (including computer software and chromatograph), connect the mobile phase pipeline, and connect the detection system.

4. Enter the main menu of the HPLC control interface and click Manual to enter the manual menu.

5. If it has not been used for a period of time, or the mobile phase has been replaced with a new one, the pump and injection valve need to be flushed first.

6. Adjust the flow rate, the first time you use a new mobile phase, you can try the pressure first, the larger the flow rate, the greater the pressure, generally not more than 2000.

7. Design distortion methods.

8. Injection and post-injection operation.

9. When shutting down, turn off the computer first, and then turn off the liquid chromatography.

10. Fill in the registration book and sign it by the person in charge.

11. The chromatographic purity of the mobile phase should be used, and the water should be 20m deionized water.

12. The column is very fragile, and the first way to do it is not to let the liquid pass through the column.

13. All liquids passing through the column need to be strictly filtered.

14. The pressure should not be too much, preferably not more than 2000 psi.

I think first of all, you should look at the retention time. The premise of increasing the peak area is to ensure that the retention time does not move back and forth. Otherwise, the focus should be on the mobile phase.

If retention time is excluded, there are several possibilities.

1.Residue at the sample port. I don't know if your instrument is filled manually or automatically.

You can wash the inlet with one needle and then one blank needle. Look for any residue of the blank needle at the retention time of the main peak. It may be that the sample of the previous needle was not washed and left for the next needle.

2.Solvent volatilization. This is more common when the solvent is pure methanol or pure acetonitrile. But what should change is not particularly obvious. You can try cryopreservation, such as a sample freezer, or simply put it in the freezer and inject it when the time is up.

3.Sample degradation. If your so-called sample is a principal component, then this should probably not exist.

If it's the impurity peaks that are getting bigger and bigger, that's possible. The largest principal component degrades, and then degrades into these small impurity peaks. As a result, these impurity peaks are getting larger and larger.

You can analyze the structure of the principal component of the sample to see if it is stable. If this is really the reason, then there is no way, or you should consider changing the solvent and cryogenic storage. Otherwise, the sample can only be used temporarily.

1. See if the precision in the following method fluctuates, and check whether your peak area increase is within the range;

2. Check the stability, whether the solvent volatilization during injection causes the sample concentration to increase;

3. If it is a fully automatic liquid phase, see whether the needle washing procedure is added;

4. After entering a needle, enter a blank needle to see how much residue there is, if the residue is large, it is recommended to add a blank between the two needles.

Suppose we want to detect a hydrophilic substance, if the mobile phase has a high proportion of water, then it is not easily adsorbed by the column, so the separation is not good. In this case, it is necessary to consider reducing the proportion of water. For pH, I use it to verify the ratio of the mobile phase formulation, for example, the standard is 9, if it is 11, there may be a problem with the mobile phase.

The learning and training column of "Chromatography World" has a detailed chromatography theory knowledge system, you can check it out.

Are you referring to the flow to the moving phase degassing, or the source instrument exhaust?

The main purpose of both is the same, both are to prevent bubbles from entering the instrument. If air bubbles enter the pump, it can clog and the instrument fails. Columns can also become clogged and lead to scrap. Air bubbles enter the detector, which affects the detection.

Mobile phase degassing is the removal of gases dissolved in mobile phase liquids. The purge venting of the instrument is to remove the gas from the solvent filter head to the front line of the pump.

The mobile phase BAI used for HPLC must be pre-deducated

gas, otherwise it is easy to be in zhi

Bubbles escaping from the system, DAO

Affects the operation of the pump. Bubbles will also be shadowed.

The separation efficiency of the plate column affects the sensitivity of the detector, the stability of the baseline, and even makes it impossible to detect. (Increased noise, unstable baseline, sudden jumping). In addition, oxygen dissolved in the mobile phase may react with the sample, the mobile phase, and even the stationary phase (e.g., alkyl amines).

Dissolved gases can also cause changes in solvent pH, which can lead to errors in separation or analysis results.

Commonly used degassing methods are: heating and boiling, vacuuming, ultrasonic, helium blowing, etc.

After the instrument is used for a period of time, the components will age, and the performance indicators of the instrument will definitely be worse than when it was first installed.