Appeal TLC and HPLC results are inconsistent

You must be a device problem, maybe the UV lamp you're using is dual-wavelength. If HPLC is detected by ultraviolet, the UV range is basically 200-600 before HPLC is done, and then the HPLC detection wavelength is adjusted to the highest absorption of UV after the maximum absorption is determined.

It stands to reason that the UV VIS Lamp detection is based on the same principle as the monitor in HPLC, and there should not be a large difference in absorption intensity. I don't know if you ran the sample and it wasn't contaminated, the best way to check the agent is to run a blank board, and then look under the UV and see if it is not absorbed. And for the HPLC you use, you see if you use the inverted HPLC, the fixed direction used for reverse elution is C18, if it is reverse elution, the order of the front is naturally reversed.

There is also an ultimate detection method Haha, I don't know if there are many double bond conjugations in the sample you are studying, if there is a lot of fluorescence absorption, run the FL to see if the root ultraviolet effect is the same, and you can verify the UV VIS results

First, it is necessary to determine what is the maximum absorption of substances A and B under ultraviolet light, and secondly, what is the content of A and B. According to your situation, it can be roughly analyzed that the absorption intensity of substance A is much greater than that of substance B at 254nm, and the concentration of substance B is much greater than that of substance A.

Under TLC, A is a trace substance, the resolution accuracy is not high, and the absorption is almost invisible, while the absorption of substance B is not large, and the single victory is large in concentration, which can be resolved.

Under HPLC, substance A is enriched in the separation column, and the peak emission is naturally strong due to large absorption, while the peak emission is weak due to the large concentration of substance B, although the concentration is large, the absorption is not strong, so the peak emission is weak.

It is recommended to do a DAD and find a wavelength with a correction factor of 1 to measure HPLC. Alternatively, it may be easier to use a refractive index detector for analysis.

Different detection equipment has different sensitivity to the same substance. Which test to use depends on what A and B are.

HPLC High Performance Liquid Chromatography (UV) UV UV-VIS Spectrophotometry GC Gas Chromatography (TLC) Thin Layer Chromatography.

What kind of product is yours, these instruments, the abbreviation of the test items.

Qualitative is OK, that is, exclusivity, but quantitative is certainly not.

When detected by HPLC, the standard can be dissolved in the mobile phase, but the mobile phase can basically only be chromatographically graded, and the main difference between TLC grade and chromatographically pure and HPLC grade reagents is the difference in purity.

【】Hello student wangchan172! The answer to your question is as follows: tlc:

Thin layer chromatography. A suitable stationary phase is applied to a glass plate, plastic or aluminum substrate to form a uniform thin layer. After spotting, the specific shift value (RF) is compared with the specific shift value (RF) of the chromatogram obtained by the same method with the appropriate control substance, which is used for drug identification, impurity detection or content determination.

Thin layer chromatography is an important experimental technique for the rapid separation and qualitative analysis of small amounts of substances, and is also used to track the progress of reactions. PC: Paper chromatography.

It is a method that can be used to test the purity of substances and separate mixtures, as it can be analyzed quickly and with few restrictions on the material, so it is an extremely convenient and useful analytical method. Paper chromatography uses the same separation principle as thin layer chromatography, where species are distributed in a stationary and mobile phase. The stationary phase is usually a filter paper on which the mobile phase will flow with the substance, and the device will separate the mixture based on the adhesion of the different species to the stationary phase and the solubility of the mobile phase.

When analyzing pigments, if the pigment contains more than one substance, the different colors of the substances will be separated according to the polarity of the solvent and the different solutes, because different molecular structures are most likely to have different polarities. These different polarities result in different solubility of solvents, so that various solutes will precipitate on the stationary phase at different locations of solvent diffusion and appear as spots, which can be analyzed according to the location and size of the spots on the stationary phase. hplc:

High performance liquid chromatography. It is an important branch of chromatography, using liquid as mobile phase, using a high-pressure infusion system, pumping a single solvent or mixed solvent and buffer with different polarity into a 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. gc:

Gas chromatography. It can be divided into gas-solid chromatography and gas-liquid chromatography. Gas-solid chromatography refers to the chromatographic separation method in which the mobile phase is a gas and the stationary phase is a solid substance.

For example, activated carbon, silica gel, etc. are used as stationary phases; Gas-liquid chromatography refers to the chromatographic separation method in which the mobile phase is a gas and the stationary phase is a liquid. For example, the inert material diatomaceous earth is coated with a layer of squalane, which can separate and measure trace amounts of methane, acetylene, propylene, propane and other impurities in pure ethylene. Good luck with your exam!

They are all methods of instrumental analysis, followed by high performance liquid chromatography, ultraviolet and visible light, gas chromatography, thin layer chromatography (this is not instrumental analysis), if you are studying analytical chemistry or refining direction, then you will use these instruments every day in the future! If you don't know what to ask!

1. System composition and working principle of high-performance liquid chromatograph.

The separation of mixtures is based on the differences in the physical and chemical properties of the components, and GC mainly uses the differences in the boiling point, polarity and adsorption properties of substances to achieve the separation of mixtures. After vaporization in the vaporization chamber, the sample to be analyzed is brought into the column by an inert gas (i.e., carrier gas, generally N2, HE, etc.), which contains a liquid or solid stationary phase, and each component tends to form a partition or adsorption equilibrium between the mobile phase and the stationary phase due to the different boiling points, polarity, or adsorption properties of each component in the sample. However, due to the flow of the carrier gas, this equilibrium is actually difficult to establish, and it is precisely because of the flow of the carrier gas that the sample components are repeatedly distributed or adsorbed and deattached in motion, and as a result, the components with large concentrations in the carrier gas flow out of the column first, and the components with small concentrations in the stationary phase flow out after flowing.

As soon as the components flow out of the column, they enter the detector, which is able to convert the presence or absence of sample components into an electrical signal proportional to the amount or concentration of the component being measured, which, when amplified and recorded, contains the full raw information of the chromatogram. In the absence of component elution, the chromatogram is recorded as the background signal of the detector, i.e., the baseline of the chromatogram.

Gas chromatography (gas

chromatography

Abbreviated as GC) is a type of chromatography. There are two phases in chromatography, one is the mobile phase and the other is the stationary phase. If a liquid is used as the mobile phase, it is called liquid chromatography, and if a gas is used as the mobile phase, it is called gas chromatography.

Gas chromatography can be divided into two types due to the different stationary phases used, which is called gas-solid chromatography with solid adsorbents as stationary phase, and gas-liquid chromatography is called gas-liquid chromatography with a monomer coated with stationary liquid as stationary phase.

According to the principle of chromatographic separation, gas chromatography can also be divided into adsorption chromatography and partition chromatography, in gas-solid chromatography, the stationary phase is the adsorbent, the gas-solid chromatography belongs to adsorption chromatography, and the gas-liquid chromatography belongs to the distribution chromatography.

According to the form of chromatographic operation, gas chromatography belongs to column chromatography, which can be divided into two categories: general packed column and capillary column according to the different thickness of the chromatographic column used. Generally, the packed column is to pack the stationary phase in a glass or metal tube with an inner diameter of 2 6 mm. Capillary columns can be divided into two types: hollow capillary columns and filled capillary columns.

The hollow capillary column is to coat the fixative solution directly on the inner wall of the glass or metal capillary with an inner diameter of only millimeters, and the filled capillary column has only been developed in recent years, it is to load some porous solid particles into thick-walled glass tubes, and then heat and draw them into capillaries, generally with an inner diameter of millimeters.

In practice, gas chromatography is mainly based on gas-liquid chromatography.

How does gas chromatography work:

Using the different partition coefficients of each component in the sample between the gas phase and the stationary liquid phase, when the vaporized sample is brought into the chromatographic column by the carrier gas to run, the components are repeatedly distributed between the two phases, due to the different adsorption or dissolution capacity of each component in the fixation, so the running speed of each component in the chromatographic column is different, after a certain column length, they are separated from each other, leave the chromatographic column in order and enter the detector, and the generated ion flow signal is amplified, and the chromatographic peak of each component is depicted on the recorder.

No, TLC stands for thin layer chromatography, and the reference used for thin layer chromatography may have problems such as low purity and many impurities.

No, the purity of the standard required by thin layer chromatography and high performance liquid chromatography is not the same, if the reference substance has a lot of impurities, it is not pure, and it is easy to damage the column, and it can be communicated.

If you have money, you can do HPLC, and if you are sure, you can only do TLC.

If you do drug standards, you can't; If you do research, you can publish articles.