1. Lecture – 1
GEB 308
Summer 2016

2. Definition of Chromatography
IUPAC (International Union of Pure and Applied Chemistry) Definition: “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.” General Definition: Chromatography is a scientific discipline (scientific field) that investigates formation, change, and movement of concentration zones of analyte chemical compounds of a studied sample in a flow of mobile phase with respect to solid or liquid stationary phases or particles.

3. A Brief History on Chromatography
The concept of separating sample components in a column was first developed in 1903 by a Russian Scientist named Mikhail Tswett (or Tsvet), who introduced the term chromatography in He is generally referred to as the father of chromatography. He invented adsorption chromatography to separate plant pigments, using a hydrocarbon solvent and a carbohydrate powder as stationary phase. The separation of colored bands led to the name chromatography. Chromatography lay dormant until Tswett’s methods were applied by some German scientists, beginning in 1931, and gradually adsorption chromatography became an established tool in biochemistry.

4. A Brief History on Chromatography

5. A Brief History on Chromatography
In the period between 1906 and 1952 there were some developments of importance. First, there was partition chromatography (liquid–liquid partition chromatography and gas–liquid partition chromatography); for which two scientists Martin and Synge won the Nobel prize in Partition chromatography and its sequential development occurred during the 1940s–1960s period. Thin layers of silica gel were introduced as an alternative of paper, which established the field of thin-layer chromatography (TLC) during late 50s. The later developments in the 1970s–1980s led to HPLC, UHPLC, and associated techniques with chromatography including UV/IR spectrophotmetry, MS/MS, NMR, etc.

6. A Brief History on Chromatography
To date, HPLC (High Performance Liquid Chromatography) has become the dominating chromatographic technique, with capillary GC (Gas Chromatography) being second only to it (for the more volatile analytes). Both GC and HPLC are mature separation techniques today; however, HPLC is still being developed toward faster and more efficient separations and also partially toward miniaturized columns, particularly for applications in the life science area. The majority of the other techniques of chromatography are in much lesser use today.

7. Basics of Chromatography
In a chromatographic system, the sample is introduced in a small volume at the inlet of a column or another carrier of the stationary phase. The mobile phase transports the sample components in contact with the stationary phase throughout the column. Due to different interactions between the sample components and the stationary phase, the sample components migrate through the system at different speeds and elute from the column with different retention times.

8. Mobile and Stationary Phase
The sample components (solutes) can usually interact directly with components of the mobile phase. When the stationary phase is a solid, often with polar surface groups, and the mobile phase is either a gas (in GC) or an organic solvent (in LC), the separation principle is based on adsorption, and the term adsorption chromatography can be used. In gas chromatography, the stationary phase can also be a liquid, where the separation principle is based on partition between the two phases. This was also the case formerly in liquid chromatography, but after the introduction of chemically bonded stationary phases into HPLC, the stationary phase cannot be described as a liquid anymore.

9. Basics of Chromatography

10. Basics of Chromatography
A solution containing solutes A and B is placed on top of a column packed with solid particles and filled with solvent. When the outlet is opened, solutes A and B flow down into the column. Fresh solvent is then applied to the top of the column and the mixture is washed down the column by continuous solvent flow. If solute A is more strongly adsorbed than solute B on the solid particles, then solute A spends a smaller fraction of the time free in solution. Solute A moves down the column more slowly than solute B and emerges at the bottom after solute B.

11. Basics of Chromatography
The retention time is defined as the time between the sample introduction and the elution from the column. At the end of the column, a detector provides a signal for all eluting components (universal detection) or for a limited number (selective detection). In a sample with many components, some compounds will coelute, partly or completely, depending on the complexity of the sample and the peak capacity of the column. With mass spectrometric detection, even coeluting components can be identified.

12. Basics of Chromatography
At any given time, a particular analyte molecule is either in the mobile phase, moving along at its velocity, or in the stationary phase and not moving at all in the downstream direction. The sorption-desorption process occurs many times as the molecule moves through the bed, and the time required to do so depends mainly on the proportion of time it is sorbed and held immobile. When the stationary phase is a solid, often with polar surface groups, and the mobile phase is either a gas (in Gas Chromatography) or an organic solvent (in Liquid Chromatography), the separation principle is based on adsorption.

13. ABsorption vs ADsorption

14. Basics of Chromatography
Schematic representation of the chromatographic process.

15. Basics of Chromatography
In the first one, the mixture of analytes A and B is introduced to the bed in as narrow a zone as possible. The mobile phase, flowing from left to right, carries them along the bed. Since all the molecules of a particular analyte do not encounter the exact same local environment in the stationary and mobile phases, the peak for that analyte will have a finite width in the chromatogram. The width of the peaks increases with the length of time they remain in the bed. Because analyte A has a greater affinity for the mobile phase, it spends more time in the mobile phase and travels faster and elutes before analyte B. Thus A and B become separated.

16. Types of Chromatography
Chromatography is divided into categories on the basis of the mechanism of interaction of the solute with the stationary phase:
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
Affinity chromatography

17. Chromatogram
When the sample components are separated and detected by a detector connected to the outlet of the column and the signals from the detector are visualized as a function of time, a chromatogram is obtained. A chromatogram is a graph showing the detector response as a function of elution time.

18. Retention Factor
At any given time during the migration through the system, there is a distribution of molecules of each component between the two phases: ns / nm, where ns and nm are the number of molecules in the stationary and mobile phases, respectively, at a given time. When ns is much larger than nm, the migration is very slow and the analyte elutes with high retention. k = ns / nm is called the retention factor. k is the recommended symbol by IUPAC for describing the retention of a compound; it is independent of flow rate, column dimensions, and so on.

19. Retention Factor
If one component migrates through the column in the mobile phase only, with no interactions with the stationary phase, the migration time is called tM . An analyte with interactions with the stationary phase will be retained and will elute at tR: tR = tM + tMk = tM(1+k) The tM can be determined by injecting a component known to have no interactions with the stationary phase. From the previous equation, k can be measured:

20. Retention Factor
Retention volume, VR, is the volume of mobile phase required to elute a particular solute from the column. The constant flow of the mobile phase is denoted as F. Now, Retention volume represents the volume of mobile phase that flowed during a specified operating time t. So, retention volume, VR = tR x F; Thus, Time units can also be replaced with volume units: VR = VM + VMk = VM(1+k) Another way to express the equation is: VR = VM + KcVs Here, Kc is a partition coefficient (Kc = [A]S / [A]M) and Vs is the stationary-phase volume.