Separation complex mixtures

By connecting a gas chromatograph to a suitable mass spectrometer and including a data system, the combined method of GC/MS can be used routinely to separate complex mixtures into theii individual components, identify the components, and estimate their amounts. The technique is widely used. 

The primary limitation of an HPLC/MS approach when compared to GC/MS is its lower separation efficiency. The separation efficiencies of GC/MS capillary columns can be in excess of 100,000, while those of HPLC are on the order of 10,000 to 40,000 thus, HPLC has a lower ability to separate complex mixtures. Because HPLC is generally utilized to separate compounds not possible via GC/MS, it provides complementary rather than competitive data. 

As yet, the number of applications is limited but is likely to grow as instrumentation, mostly based on existing CE systems, and columns are improved and the theory of CEC develops. Current examples include mixtures of polyaromatic hydrocarbons, peptides, proteins, DNA fragments, pharmaceuticals and dyes. Chiral separations are possible using chiral stationary phases or by the addition of cyclodextrins to the buffer (p. 179). In theory, the very high efficiencies attainable in CEC mean high peak capacities and therefore the possibility of separating complex mixtures of hundreds of... 

Two-dimensional GC can be used to separate complex mixtures of polyaromatic compounds, and MS used to subsequently identify the compounds. In this method, the original sample is injected into a gas chromatograph with one type of column. As the components exit the first GC, they are fed into a second GC, with a different column, for further separation and finally into a mass spectrometer. In this way, compounds that coeluted from the first column are separated on the second. Focant et al. [19] were able to separate polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), and coplanar polychlorinated biphenyl (cPCB) using this type of analytical procedure, including isotope dilution TOF-MS. These compounds are frequently found as contaminants in soils surrounding industrial settings thus, the ability to separate and identify them is extremely important [6,12,19],... 

As discussed in Chapter 7, gas chromatography (GC) is used to separate complex mixtures of volatile organic compounds. However, unless pure authentic standards are also analyzed to compare retention times, it is not possible to identify the components by GC alone. However, by connecting the output of a GC to a mass spectrometer, and by removing the carrier gas to maintain the low pressures required, it is possible to both separate and identify these complex mixtures. This method is the gold standard for the identification of organic samples, if they are sufficiently volatile. 

Tell how the temperature programming feature of most modern GCs can be useful in separating complex mixtures. 

A flexible and efficient chromatographic separation, offering the possibility for separating complex mixtures of compounds with a range of polarities, was needed. This preferably should be accomplished without tedious derivatisation steps, and should allow direct analysis of aqueous samples. 

The distillation technique is not used to separate complex mixtures, but finds its acceptance more for the preparation of large quantities of pure substances or the separation of complex mixtures into fractions. The technique depends on the distribution of constituents between the liquid mixture and component vapors in equilibrium with the mixture two phases exist because of the partial evaporation of the liquids. How effective the distillation becomes depends upon the type equipment employed, the method of distillation, and the properties of the mixture components. The distinguishing aspects of distillation and evaporation are that in the former all components are volatile, whereas in the latter technique volatile components are separated from nonvolatile components. An example of distillation would be the separation of ethyl alcohol and benzene. An evaporative separation would be the separation of water from an aqueous solution of some inorganic salt, for example, sodium sulfate. 

Thin-layer chromatography (TLC) is a common laboratory technique for separating complex mixtures of solutes, usually by an adsorbtion mechanism. Several laboratories have applied the technique to the separation of polymer fractions and characterization of polymer molecular weight distributions. This work reviews the experimental results and theoretical approaches to the fractionation mechanisms. 

Analytical methods used to identify monomers have improved significantly from those that quantify whole classes of compounds, such as amino acids, peptides, proteins, and primary amines (Undefriend et al., 1972) or carbohydrate-like compounds (Johnson and Sieburth, 1977) to ones that are molecule-specific (Table II). Most of these methods are based on combining chromatography techniques that can separate complex mixtures of molecules with highly sensitive detectors that can approach the nanomolar or picomolar range. Monomers are usually present at low concentrations, so... 

There are many well-established methods for separation and structure determination of ecdysteroids.20 27 A newly described method is two-dimensional thin-layer chromatography. It has been used to separate complex mixtures of phytoecdysteroids. Silica plates were developed first with toluene—acetone—ethanol—25% aqueous ammonia (100 140 32 9 v/v) and then developed in the other direction with chloroform—methanol—benzene (25 5 3 v/v)48... 

MD (multistage, multicolumn) chromatography was early discovered to be a powerful tool for separating complex mixtures [8]. Two of the protagonists were J. C. Giddings [9,10] and J. F. K. Huber [11]. MD-LC is based on coupling columns in an online or offline mode, which are operated in an orthogonal... 

It was during the 1970 s and early 80 s that advances in technology transformed natural product discovery programmes. The advent of HPLC, and later coupling to UV diode-array detectors and then mass spectrometers, improved the efficiency of dereplication procedures. The ability to rapidly separate complex mixtures reduced the time from lead identification to natural product structure. The discovery of doramectin is a fine example of the use of such technology. 

A linear gradient generated like this is suitable for many appli- cations. However, for separating complex mixtures of oligonucleotides a very complicated gradient may be required. It may also be necessary to make empirical refinements of the gradient after the initial run of a complicated mixture of oligonucleotides. 

Gel Permeation Chromatography This is again a potentially valuable technique since it can, in principle, separate complex mixtures of macromolecules directly on the basis of molecular size. Indeed, in cases such as proteins, with molecules of very different sizes, and where appropriate calibration standards are available, it is possible to obtain well resolved, chemically pure fractions and to determine molecular weights directly. There is, however, the possibility of artefacts arising due to interaction with the gel [23y24]. This may be irreversible that is sample material may simply be retained on the top of the gel column, or it may be more subtle, in which case the retarded fractions are eluted, but later than would be expected solely on the basis of molecular size. 

When using 2D electrophoresis to separate complex mixtures of proteins, why is it necessary to perform isoelectric focusing in the first dimension ... 

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