Basic Chromatography基本的色譜法

1、Chromatographic methodsChromatography is the science of physical separation of species in a mixture. It may be used to measure components of both qualitatively and quantitatively.The basis of all chromatographic techniques is the principal of retention. That under certain conditions molecules move a

2、t different rates through a separating bed.When to use Chromatography:To measure many different compounds in complicated multi-component mixtures.AnalytesPesticides, dioxins, PCBs, PAHs, benzene, hydrocarbons, chlorofluorocarbons, c.f. SpectroscopyUsed for simple mixtures, or very specific measureme

3、ntsMatricesSoil/sludges, water, solvents, biological fluids, air, natural gasThe most known chromatographic process is when a spot of ink on wet paper spreads out to leave bands of colours which made up the colour black. The different colours of ink travel at specific rates across the wet paper depe

4、nding on the degree to which each colour sticks to the surface of the paper. In chromatographic terms the paper is known as the stationary phase and water, the mobile phase, two elements common to all chromatography. Any substance exposed to two such media will partition itself between them dependin

5、g on its affinity to each phase. Molecules with a high affinity to the mobile phase will move more rapidly than one with high affinity to the stationary phase. In modern chromatographic techniques the system is housed within a column. Stationary phases either exist as an interior coating within the

6、column or on the surface of a porous packing material. The type of stationary and mobile phases can be changed to make the system as efficient as possible for separating each mixture.The particles used to pack columns are commonly porous silica beads of 3-10 m diameter.Columns used have internal dia

7、meters of 50 m up to 1 cm.The silica of both packed and coated columns is highly porous containing immense matrices of channels.This creates massive surface areas of square metres per gram.Pores on surfaceClose packed beads Inside the column, the sample exchanges between the mobile and the stationar

8、y phase. Analytes that interact more strongly with the stationary phase and more weakly with the mobile phase tend to take longer to pass through the column. An equilibrium between the two phases is set up. Molecules in the mobile phase continue to move down the column interacting with the stationar

9、y phase. The mobile phase moves on to form a new equilibrium with the remaining molecules on the stationary phase to re-establish the equilibrium. As bands move down a column those with a higher affinity to the stationary phase are retained. Also note that the bands spread out.timemobile phaseflowst

10、ationary phase particleweakly retained analytestrongly retained analyte When the stationary phase interacts with retains a compound it is said to have a particular capacity factor (k)phasemobileinmolesphasestationaryinmolesk If a compound is unretained by the stationary phase it will move at the sam

11、e rate as the mobile phase.If an unretained compound elutes from the column after time t0 and a retained compound after time tr then k can also be expressed by:00tttkrThe massive surface area of the silica in Liquid Chromatography can be coated with many different compounds which interact with passi

12、ng analytes. There are many different types of interactions which cause retention including dispersive, polar, ionic and size-exclusion.As a result there are hundreds of different stationary phases on the market and unlimited mobile phases which can be made in the lab. Detectors in chromatography gi

13、ve a response signal for the concentration of eluting analyte and the resulting chromatogram is a plot of response against time. Ideally, each peak corresponds to an single analyte. The time of elution of a peak tells us the identity of the analyte. The height or area of the peak is related to the c

14、oncentration of the analyte making the analysis quantitative. This is if the peak is due to ONE eluting analyte, and if adjacent peaks are resolved from each other. If two analytes have similar retention times their peaks will overlap, what can we do to totally separate or resolve them?Increase the

15、difference in retention.Make the peaks narrower. If a chromatogram has narrow peaks then the column on which the analytes were separated is said to have a high efficiency. The measure of column efficiency is the parameter plate height or H, the lower the plate height the more efficient the column.21

16、6wtLHr Where w is the width of the peak, tr the retention time and L the length of the column.Chromatography - DistillationEach separate level represents another equilibrationThe more levels the better to separationI.e. the lower the plate height the better! We know peak width broadens as the analyt

17、es pass through a column which explains the relationship between column length and efficiency. The length of a column is a fixed value so how can we alter efficiency. What are the causes of broadening of an analyte band. What are the factors which affect the process of band spreading? 216wtLHr Low p

18、late heights H are achieved from a short column length L and a large retention time : peak width ratio. There are said to be three modes in which band broadening can take place within a column. The parameter which has a direct effect of all of these is the length of time the component spends in the

19、column, which is reliant on the velocity of the mobile phase. Parameter B is broadening through simple diffusion from a point with time, so as time spent in the column decreases the efficiency increases. Parameter Cb broadening due to the different lengths of path molecules in a band must take throu

20、gh the porous stationary phase. Parameter Cm is broadening due to time taken for equilibrium to be reached between the stationary and mobile phases. The overall result of these three broadening effects is the van Deemter curve. The lowest point in this curve relates to the optimum mobile phase veloc

21、ity for an efficient analyte band.van Deemter Curves. Efficient peaks are easy to resolve but at what point can we say that two peaks are sufficiently resolved for analysis? For two peaks A and B the resolution between the two can be expressed by an equation: BAArBrwwttR2 If resolution R calculated

22、through the retention times and peak widths of the two peaks is equal or greater than 1.5 then the peaks are said to be resolved.If two different mixtures both containing a component A are separated on the same column using the same mobile phase, the retention time of A will be the same for both sep

23、arations.Using this knowledge standard mixtures are used to identify unknown peaks from a separated mixture.Standard mixtures can be purchased for many mixtures of compounds.The known concentrations of components in standard mixtures also allows us to determine concentrations of the analytes in our

24、samples. This standard mixture for the hydrocarbons found in air is overlaid on an actual sample chromatogram in red. Another way to determine the identity of a peak is to spike your sample with an excess of an expected compound. The increase in peak size after spiking a sample will identify the ori

25、ginal peak. Here the sample was spiked with compound X and it is obvious which is the corresponding peak. There are many different types of chromatography which have been developed to separate different types of mixtures. So far we have discussed mobile phases which instinctively we imagine as liqui

26、ds. In gas chromatography (GC) the mobile phase is a gas such as Helium or Hydrogen. In GC the mobile phase does not have much effect on the analyte equilibrium, we can see for the hydrocarbons in air sample, retention is highly dependant on molecule size or volatility.Types of chromatography GC columns are usually have coated walls instead of packed particles. The dimensions of GC colum