Chapter 26 – Chromatographic Separations

Invented by Russian Botanist Mikhail Tswett in 12+ Nobel Prizes have been awarded for work in which chromatography played a vital role.

Stationary Phase: Mobile Phase:

A large number of molecules start migrating - each moves in a direction in one step of equal length, then another, etc. Eventually, they will be dispersed. The flow of the __________________________ keeps the molecules flowing towards a _________________ as a ___________________. What Determines Elution Order? GC In gas chromatography, elution order is generally determined by:

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• What will elute first? What will elute last? What won’t be eluted at all? What Determines Elution Order? HPLC

• What is most likely to have an affinity to a non-polar stationary phase? What is the least likely? How does this determine elution order?

Theory of Chromatography Analyte is in either the SP or MP at all times. An equilibrium exists (at constant If c = the concentration of analyte; K is the equilibrium constant, called the Distribution Constant or Partition Ratio

Retention time, The time it takes the analyte to travel (in the MP) through the SP to reach the detector. Dead time (volume), The time it takes the analyte’s solvent to travel through the SP, unretained.

Retention Factor also called capacity factor A number that approximates how well an analyte has been retained Ideally, kA’ is between We can control experimental variables to optimize this. Selectivity Factor For two components of a mixture, this tells us how selectively the column can separate the two from each other. This number relates to the ___________________between two peaks. Width of Peaks If separation is complete, each peak represents a component of the mixture. The width of the peak, W (measured in time units) is dependent on how well the molecules of one component are traveling together in the column.

Chromatographic peaks should have a . _______________________ or ____________________ are signs that there is a problem with your experimental conditions. Problems that can cause tailing in HPLC Problems that can cause fronting in HPLC Ideal Peak Width Keep your peaks as Gaussian (symmetrical) and_______________ as you can for good quantitation (____________________ the area under the peak). This means, in addition to avoiding the aforementioned problems, you want your chromatographic run to be as _______________ as possible for quantitation.

Void Volume Chromatography trouble-shooting is all about plumbing. You want as little _________________________ as possible, and no areas of increased volume where molecules can spread out and ___________________ the peak. If you are not careful with your plumbing, you can accidentally leave these areas, leading to void volume. Your chromatography will suffer. Active Sites This is specifically related to HPLC columns. If your column is poor quality or has degraded, this problem could arise and cause peak tailing. Active sites are acidic, so __________________components will suffer tailing more than neutral or acidic components. Plate Theory A chromatographic column is comparable to a distillation column with ____ plates. The ________________ plates, the better your distillation - your distillate is better separated from other components in the mixture with _______________ __________________ ______________.

Theoretical Plates

Height of Theoretical Plates It is comparable to the distance between two plates in the distillation column – the ______________________ the distance, the better your separation. H and N are numbers that describe a particular column. The way the column is designed determines the values for these figures of merit. Each _____________________ that is separated has an ____ value. A good column is considered to be an _________________ column.

Van Deemter equation Predicts the maximum _____________________ of a column, which depends on the flow rate (________________) of the mobile phase.

Getting the Best Separation

____________________ describes how well two components are separated (resolved) from each other.

General Elution Problem

Homework: Chapter 26 #14 – 22, 23 b-i