HIGH-PERFORMANCE LIQUID CHROMATOGRAPH (HPLCs)
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The HPLC is a master specialist in determining the identity and quantity of elements and molecules, such as the activity in an herb, the product of an enzymatic reaction, or any molecule which absorbs light or that can be made chromophore (able to absorb/transmit light). The HPLC is very specific in being able to absolutely determine what a substance is and exactly how much of it there is.
HPLC works by automatically injecting a small volume of liquid sample into a column packed with particles 1⁄20 the thickness of a white sheet of paper. The liquid sample is forced through the column by powerful micropumps. The detector sends a digital signal to the computer, where specialized software is used to identify and determine the quantity of the separated components.
We use it routinely to analyse the composition of compounds present in complex mixtures, such as water- and fat-soluble vitamins. We also use the HPLC to analyse a large variety of plant material, for example astragalus, dandelion, and red clover.
This is a typical liquid chromatogram of water-soluble vitamins; these are called chromatographic peaks, and each one represents a separated compound.
Additional info (found in other website labs) Do not plagiarize 🙂 but edit later
HPLC Mass Spectrometry as an analytical tool
High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an ideal tool for the identification, quantitation, and mass analysis of components, often used to determine the chemical composition and purity of chemicals. HPLC-MS has high sensitivity and is optimal for performing precise and reproducible quantitative analyses. The method is ideal for detecting heat-labile compounds, such as proteins and vitamins in food products. Food contaminants are also often quantified using HPLC-MS.
In HPLC-MS, the two techniques (HPLC and MS) are connected by an interface that transfers the separated components from the liquid chromatograph column into the mass spectrometer ion source. An interface is needed since HPLC operates at high pressure and the MS system has a high vacuum.
HPLC includes a mobile phase and a stationary phase, both of which can be modified to suit the sample matrix and the desired properties to be determined. Usually, the mobile phase is adjusted to suit the sample, and the stationary phase is adjusted to work well with the mobile phase. The degree of compound separation is based on the compound’s affinity for the mobile phase.
HPLC methods can be divided into two main categories based on the properties of the stationary and mobile phases. A combination of a polar stationary phase and a non-polar mobile phase is called “normal-phase chromatography” and the opposite of that, the combination of a non-polar stationery and a polar mobile phase is called “reverse-phase chromatography”.
Normal-phase HPLC
In normal-phase HPLC, the column is usually filled with silica particles. The silica is polar and will bind to polar molecules in the mobile phase. This means that the least polar compounds elute first and the most polar compounds last. Normal-phase HPLC is suitable for highly hydrophobic or hydrophilic compounds, compounds that are not soluble in water, and compounds that may decompose in water. The ideal use of normal-phase HPLC is the separation of isomers.
Reverse-phase HPLC
In reverse-phase HPLC, the stationary phase usually consists of C8 or C18 silica (silica derivatized with alkyl chains). Unlike in normal-phase HPLC, in reverse-phase HPLC, the most polar compounds elute first and the least polar compounds last. The stationary phase can be composed of different solids to meet different needs.
Combining HPLC with Mass Spectrometry
Once compounds have been separated using HPLC, they are identified and their contents are determined by mass spectrometry (MS), which creates a mass spectrum that is unique for every compound. In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios.
It is also possible to combine HPLC with detectors other than the mass spectrometer. In HPLC-DAD analysis, for example, a diode-array detector is used instead. The choice of the most appropriate detector depends on the analyte and the goal of the analysis. In general, HPLC-MS is more suitable for identifying unknown components, while the DAD-detector works well in quantifying known components.
Samples and sample preparation
HPLC-MS is suitable for samples in liquid form. For solid sample matrices, sample preparation is necessary. Compounds that are being determined must be extracted into a solvent and filtered before analysis.
