Intro to Chromatography

Chromatography In chromatography, a physical separation of a mixture occurs. The mixture is poured or enters a column (a hollow cylinder). The column may be long or short, wide or narrow depending on the need. The column contains “packing” material that the mixture then passes through.

Chromatography The packing material may be a layer on the wall of the column or the column may be completely filled with the material. The packing material is called the “stationary phase”. Stationary phases may be clays or other inorganic solids, silica, organosiloxanes, or a thin layer of liquid organosiloxanes coated on a solid support (silica).

Chromatography The packing material separates the different analytes in the mixture as different analytes travel at different speeds through or along the stationary phase. How does this happen? Simply stated, different analytes interact differently with the stationary phase.

Chromatography If the analyte interacts very weakly with the stationary phase, it will pass through the column quickly. If the analyte interacts very strongly with the stationary phase, it will be retained by the column for a much longer period of time (perhaps forever, which is bad).

Chromatography As they travel at different velocities through the column, the analytes will exit the column at different times. The analytes have been separated.

Chromatography In order for the analytes or solutes to move through a column, there is a “mobile phase” that carries them through. The mobile phase is either liquid (LC, IC, CE), gas (GC), or a supercritical fluid (SCF usually CO2) The fluid entering the column is the eluent, while exiting is called the eluate. The entire process is called elution.

Brief History of Chromatography 1906: Chlorophyll was separated by “open-column”, or “gravity” chromatography with calcium carbonate as the solid phase and pet ether as the mobile phase. 1940’s: Partition and paper chromatography developed. 1950’s: TLC, gel filtration, gradient elution, and GC developed. Commercial HPLC developed.

Solute-Stationary Phase Interactions There are 5 main different categories of solute-stationary phase interactions: Adsorption: solute is physically adsorbed (regulated by kinetics) onto the surface of a solid stationary phase. Partition: solute interacts with a liquid stationary phase and equilibrates between the stationary and mobile phases. Ion-exchange: solute ions (cations or anions) are attracted electrostatically to ionic end groups of the solid stationary phase (resin).

Solute-Stationary Phase Interactions Size exclusion or molecular exclusion or gel filtration: the solid stationary phase has pores that exclude (too small) large molecules but allow small molecules to enter. So large molecules pass through quickly, and small molecules pass through very slowly. Affinity: a specific type of solute molecule interacts with a specific group attached to the stationary phase. Antibodies may be attached to the stationary phase to separate out a particular protein. Only a specific solute attaches. It is then washed out after all the rest of the solutes have eluted.

What We See: The Chromatogram Various types of detectors (from FID, MS, UV, RID, ECD, fluorescence, etc.) detect the solutes as they emerge from the column. A plot of the detector response vs. time gives the chromatogram of the different solutes that were detected.

What We See: The Chromatogram The peaks should be gaussian with a symmetrical shape. The standard deviation of the peak is . The time at which the solute elutes is the retention time, tr The peak width at the baseline is w and is 4 .

What We See: The Chromatogram The height of the peak from baseline to apex is h. The peak width at the half-height, 1/2 h, is called the half-height peak width, w1/2, and is 2.35 . The void time or dead time, t0, is the time it takes for the mobile phase or an unretained solute to run throught the column. It is also called the dead volume or void volume.

What We See: The Chromatogram The distance between 2 adjacent peaks is related to the resolution, R. Although we don’t want 2 adjacent peaks to be too close, or actually overlap, we don’t want them to be too far apart either (as time is solvent and money). So we want an adequate resolution, but not too high. Optimum R is ≥ 1.5.

Resolution of Solutes How do you calculate the resolution between two peaks from a chromatogram? We will talk about resolution later during the HPLC discussion.

Peak Shape: Broadening Ideally, solute peaks would be extremely sharp lines! But instead they normally have a Gaussian shape. Also, the longer the retention time, the broader the peak. Why is this? There are several factors involved.

Peak Shape: Broadening As a solute is injected into the column, regular diffusion, called longitudinal diffusion occurs. This would occur even in a hollow column with no stationary phase. This is just the phenomenon of a small plug of solute broadening as it enters a new space.

Longitudinal Diffusion As the solute particles spread out, the peak shape also broadens. For short retention times, the peak is sharper, yet still has the Gaussian distribution due to normal spreading. For longer retention times, more spreading has occurred due to the longer time, so the peak is broader. The higher the flow rate of the mobile phase, u, the shorter the retention times, so the sharper the peaks will be.

Other Factors in Broadening There are other diffusion factors that affect the width of the peak: Eddy diffusion: this is due to the fact that solute particles can take different paths through the column and stationary phase. As not all pathlengths will be equal, the solute particles will elute at slightly different times.

Other Factors in Broadening Mobile Phase Mass Transfer: this occurs when one solute particle travels very close to the stationary phase. This causes friction that slows this particle down. It would have a slightly longer retention time than a solute particle that didn’t get as close to the stationary phase.

Other Factors in Broadening Stagnant Mobile Phase Mass Transfer: this occurs when a solute particle travels between stationary phase groups and gets trapped. It then diffuses back out and continues down the column. This also lengthens the retention time.

Other Factors in Broadening Stationary Phase Mass Transfer: this is the chemical interaction between the stationary phase particles and the solute particles and how the solute particles equilibrate between the stationary phase and the mobile phase.

The Ugly-Wugly Peaks Sometimes peaks do not have normal Gaussian shapes. Common ugly peaks include tailing peaks, cutoff peaks, and double peaks.

Tailing Peaks Tailing peaks are usually caused by more polar solutes that have strong interactions with the stationary phase. A higher fraction of these solutes elute slightly later than normal, so the peak shape is distorted towards the tail end. Choosing the right column, temp, and mobile phase helps avoid this.

Frontended Peaks Frontended peaks are sort of the reverse of tailed peaks. Now a higher fraction of solute particles elute earlier than normal, so the peak shape is distorted towards the front end. This is typically caused by overloading the column with too much solute, or just injecting too much. Lower the injection volume or the solute concentration.

Double Peaks Double (or even higher multiple) peaks for a single solute is also common. For manual injection, it is just due to inexperience in the proper injection technique. But in HPLC, it may also be caused by preparing the solute in a solvent system that is “stronger” than the mobile phase.

So the solute solvent should be the mobile phase or weaker than the mobile phase, and small injections should be used.

Why Do We Do Chromatography? There are 3 reasons to do chromatography: Qualitative analysis: identify solutes present in mixture Quantitative analysis: determine the amount of solute(s) present in mixture Isolation and purification of a particular solute(s): fraction collection

Major Types of Chromatography GC (#1 or 2) HPLC (#1 or 2) IC CE

GC or LC? Which to Choose? HPLC is more universal than GC, as only about 20% of compounds can be analyzed by GC. To do GC, the solute must be volatile or it must be derivatized to a volatile substance. This means that the MW is typically below 500 amu for GC.

GC or LC? Which to Choose? For GC, the solute must also be able to survive high temperatures. If suitable, GC can separate both polar and nonpolar substances. But GC is cheap and easy with no waste.

GC or LC? Which to Choose? In HPLC, much larger molecules can be analyzed, only the solubility in the mobile phase limits. Low volatile solids and liquids are easily analyzed. Room temperature analysis is typical. Can separate polar and nonpolar compounds. Ions can also be separated with the right column (kind of cheap IC). More waste unless use ultra-low flow rates.