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    Home > Biochemistry News > Biotechnology News > Introduction to UPLC (Ultra-Efficient Liquid Chromatography).

    Introduction to UPLC (Ultra-Efficient Liquid Chromatography).

    • Last Update: 2020-10-21
    • Source: Internet
    • Author: User
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    HPLC
    With the progress of science and technology, the requirements for liquid phase
    chromatography
    technology are also increasing, and improvements from a technical point of view alone are no longer possible. This requires a re-understanding of the liquid chromatography from both scientific and technical point of view, or from a theoretical height. As a result, the UPLC (Ultra-Efficient Liquid Chromatography) concept was proposed, with the limits of HPLC as its starting point.in 1996, waters introduced Allance HPLC with the primary goal of improving the "precision" of the liquid chromatography. At the time, most companies believed that HPLC technology had reached its extremes and that there was no higher demand for performance from users, so HPLC's goal was to reduce costs and move to lower prices for wider applications.
    In response to this notion, Waters argues that HPLC technology has not reached its limits, users have higher requirements for HPLC, and that improved HPLC accuracy is of great benefit to better and more reliable results, as well as a great boost to compliance.at the forefront of science and technology in the world today, liquid chromatography users now have new needs. The first is the need to improve productivity, as a large number of samples need to be completed in a very short period of time, such as metabolomic analysis, the second is in the analysis of
    bio-chemical
    samples and natural products samples, the complexity of the samples on the separation capacity of higher requirements, and the third is in the use of MS and MS/MS testing techniques, the quality of the connection put forward higher requirements. In short, we need to "get better results faster".today, we find that with the progress of science and technology, the requirements of liquid chromatography technology are also increasing, from a technical point of view alone can not be improved. This requires a re-understanding of the liquid chromatography from both scientific and technical point of view, or from a theoretical height. Therefore, the concept of UPLC (ultra-efficient liquid chromatography) is very natural. In short, UPLC is using the limits of HPLC as its starting point.theory as early as 1956, J.J. van Deemer published his famous theory: the van Deemer curve and its equations. The earliest theory was used on gas chromatography, but it can also be applied to liquid chromatography that later appeared. Waters introduced the concept of UPLC by studying this famous equation.to explore this famous equation first. If only the relationship between theoretical tower height (H) and flow rate (line speed; u) and
    filler
    particle size (dp) is concerned, the equation can be simplified as follows: , item A represents the good degree of particle size and column bed filling, item B represents axial diffusion, and item C represents mass transmission. We can see the phenomenon shown in Figure 1 from the curve of different particle sizes:First, the smaller the particle size, the higher the effect of the column, second, each particle size has its own optimal column speed, and finally, the smaller particle size makes the highest column efficiency point move towards a higher flow rate (line speed) direction, and has a wider line speed range. Therefore, reducing particle size not only improves the efficiency of the column, but also improves the speed. The use of higher flow rates is
    by the pressure resistance
    and instrument pressure resistance of the color spectral column.
    , the high column efficiency of small particle fillers cannot be reflected without the optimum flow rate. In addition, higher column efficiency requires a smaller system volume (dead volume), faster detection speed and a series of conditions to support, otherwise the high column efficiency of small particle fillers can not be fully reflected.So to truly create a whole new field of separation science, UPLC, the following issues must be addressed: : significantly improve the performance of the column, first, to solve the pressure resistance of small particle fillers, and second, to solve the problem of small particle fillers filling, including particle size distribution and the structure of the column.
    high-pressure solvent delivery unit (more than 15000psi).
    system overall design to reduce the volume of the entire system, especially the dead volume. And solve the problem of pressure resistance and leakage under ultra-high pressure.
    automatic sampler to reduce cross contamination of the sample.
    detector, optimize the flow pool to solve the problem of high-speed detection and diffusion.
    system control and data management to solve the problem of high-speed data collection and instrument control.new chromatography fillers and filling techniques UPLC separation is only possible after the synthesis of new, pressure-resistant 1.7-particle fillers with a narrow particle size distribution. Chromatography should cover several aspects: first, the synthesis of fillers to obtain high-quality filler particles, including high-pressure,
    -acid
    and so on. The second is the screening of particles to select fillers with the narrowest particle size distribution possible. Finally, the filling technology is to ensure that the particles are blocked without causing a large increase in anti-pressure.. Waters uses hybrid particle Technology - HPT, invented in 1999, to synthesize the second generation
    organic
    silicon fillers. It uses double (triethyl silica) ethane to form bridge ethyl groups in silicone. Thus the synthetic fillers have more "cross-linking" structures inside, and their mechanical strength has been greatly improved, with pressure resistance exceeding 20,000psi.
    use this technology, Waters synthesizes fillers below 2 particles - 1.7 particles of "ACQUITY UPLCTM" fillers. In order to obtain better pressure resistance and mass transmission, the filler's hole volume and aperture are also optimized. the filling technology of traditional column fillers is affected by two aspects, resulting in the performance and quality of existing small particle filler columns are not satisfactory. First of all, its particle size distribution is generally wide, for example, 5 particles in the filler will have a large number of particles below 4 and above 6, therefore, usually use 2 sieve plate at the exit of the column to intercept the filler, to prevent its leakage.
    , if you use a sieve plate below 2, the back pressure of the sieve plate rises very quickly, even exceeding the backpressurized generated by the filler. Therefore, most of the 3.5, 2.5 or lower particle size fillers still use 2 sieve plates, only a small section 5 filler at the head of the column. Therefore, the chromatography column of the existing small particle filler is far from the theoretical or ideal state. . Waters' ACQUITY UPLCTM uses stricter screening techniques to narrow the distribution of 1.7 fillers and uses new sieve plates (patent applications) and other column hardware (pillar tubes and their connectors) to fill under pressure of more than 20,000psi. Waters has installed a new column filling line and new test equipment for this purpose. As a result, acQUITY UPLCTM columns have made a qualitative leap in performance and quality. synthesis technology of filler, particle screening technology, screen plate and color column hardware technology, filling column at higher pressure is the key to UPLC column performance and quality assurance. ultra-high-pressure liquid chromatography pump Waters has the ability to manufacture ultra-high-pressure pumps, unC's Professor Groove uses ultra-high-pressure liquid chromatography pumps specially manufactured by Waters. In addition to sealing and high-pressure power, the main problems of ultra-high-pressure chromatography pumps are the compression of solvents under ultra-high pressure and insulation heating. . ACQUITY UPLCTM is equipped with a two-way high-pressure gradient pump powered by a separate plunger and switched by four solvents, with pressure resistance of up to 15,000psi at 1 ml/min flow rate for precise and reliable gradient performance. The new ultra-high pressure infusion pump enables unique small particle filler technology to operate at optimal flow rate to give full play to its characteristics.
    integrated and improved vacuum degassing technology, so that four mobile phase solvents and two sampler needle washing solvents at the same time get good degassing. The reproducibleness of its gradient is also very good, although the retention time is within 1min, but its reproducible is comparable to the reproducible of HPLC. Automatic Sampler In order to reduce dead volume and cross-contamination, autoseedators are designed using many new technologies, such as needle sample probes (XYZZ'), pressure-assisted samples, and so on. The so-called "in-pin" XYZZ' design, in fact, is the use of liquid chromatography pipeline (PEEK material) as a sample needle to reduce the dead volume, and "outer needle" is a small section of hard pipe, used to break the sample cap. pressure-assisted samples to ensure reliable and reproducible samples at such a small instrument's system volume is a very important measure. In order to reduce cross-contamination, a strong, weak double solvent injection needle cleaning procedure is used, the two needle washing solvents have also taken degassing measures. high-speed detector , however, the high-voltage, high-speed UPLC, supported by the new column, challenges the detection of ACQUITY UPLCTM results. The first is the speed problem, in a short period of time there are so many chromatography peaks need to be faster data acquisition rate adaptation, at least above 10Hz, but also need to reduce the sample in the detection pool reside time. Of course, like ordinary HPLC detectors, the signal-to-noise ratio is the goal of new detectors. although a small detection cell (<1) had to be used to reduce diffusion, efforts were made to increase the flow and transmission of light energy to reduce noise. At the same time, faster sampling rates and very small time constant values must be used to accommodate the very narrow chromatography peaks produced by UPLC.
    ACQUITY UPLCTM uses a new fiber-guided, Teflon AF pool wall flow pool, a 10mm light range (the same as a normal HPLC) and a volume of only 500nL (one-20th of a normal HPLC). When the beam is fully introduced into the flow pool via optical fiber, the characteristics of Theflon AF are fully refracted in the pool wall without losing light energy, while the sampling rate reaches 40 points/s. detector designed in this way not only adapts to the high-speed and high-resolution characteristics of UPLC, but also greatly helps to improve UPLC sensitivity. Optimize the overall performance of the system Waters ACQUITY Ultra Performance LCTM system's overall design optimizes the ultra-low system volume and dead volume, making it necessary for a successful UPLC with all the benefits of low diffusion and high-speed inspection, while also taking full advantage of the
    Mass Spectrometrography
    electric spray ionization interface. For this reason, the ACQUITY UPLCTM system is the ideal mass spectrometretral interface. when connected to our mass spectrometrometrometromet technology, ACQUITY UPLCTM system solutions provide more sensitive LC/MS and LC/MS/MS analysis. For example, when with Micronass? When LCT PremierTM connects, the quality and speed of the data is amazingly improved. application Because ultra-efficient liquid chromatography (UPLC) is an emerging field and Waters' ACQUITY UPLCTM system is just beginning to emerge, there is still a lack of published application data. Compared with traditional HPLC, UPLC's speed, sensitivity and separation are 9 times, 3 times and 1.7 times higher than HPLC.
    upLC applications are metabolomic analysis and other bio-chemical fields. Other applications that can be thought of today include the analysis of natural products. The use of UPLC to connect to mass spectrometrators such as Tof or Q-Tof is a great boost to the analysis of natural products, especially the development of the field of Chinese medicine research.
    most bio-chemical samples and natural products are very complex, can be separated chromatography peak more will feel inadequate. Figure 3 shows
    HPLC
    hpLC and UPLC chromatography (ultraviolet detection) of the two fingerprint maps of peptides.
    Under the same conditions, UPLC can separate more than twice as many chromatography peaks as HPLC, so it is possible to further study: maintaining separation and pursuing faster analysis speeds, making more samples in the same time, or optimizing separation to separate more chromatography peaks over the same time.
    when referring to "proteomics" or "metabolomics", the difference with the absence of "groups" is that the sample size is very large and requires the analysis of thousands of samples in a short period of time. The high speed advantage of UPLC without losing the degree of separation can be fully demonstrated here.
    .
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