CHAPTER6:CHROMATOGHRAPHYGeneral Principle•Chromatography encompasses a diverse and important group of methods that allow the separation, identification and determination of closely related components of complex mixtures.• commonly used in the pharmaceutical and biotech industries, R&D, manufacturing, and quality control. Also applications in food, water, and environmental monitoring. For instance, tragedy that occurred in China in 2008 in which baby formula was found to be contaminated with melamine•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 moves in a definite direction (mobile phase). • In all chromatographic separations, the sample is dissolved in a mobile phase which may be a gas, a liquid or a supercritical fluid (a substance is heated above its critical temp which a distinct phase cannot exist, regardless of pressure.

Continue…..Two phases are chosen so that the components

of the sample distribute themselves between the mobile and stationary phase to varying degrees. (Based on establishment of an equilibrium between a stationary phase and a mobile phase.)

Those components that are strongly retained by the stationary phase move slowly with the flow of mobile phase.

In contrast, components that are weakly held by the stationary phase travel rapidly.

As a consequence of these differences in mobility, sample components separate into discrete bands/zones that can be analyzed qualitatively and/or qualitatively.

Continue…..Chromatography can be characterized by:

◦Physical means by which stationary phase and mobile phase are brought into contact column chromatography: stationary phase in

narrow tube through which mobile phase is forced under pressure

planar chromatography: stationary phase is supported on flat plate. Mobile phase moves through stationary phase by capillary action or under influence of gravity

◦Types of mobile and stationary phases and the kinds of equilibria involved in the transfer of solutes between phases. Liquid chromatography gas chromatography supercritical-fluid chromatography

Continue…..Chromatograhic processes

◦Adsorption: stationary phase is solid on which sample components is adsorbed. components distribute between two phases through a combination of sorption and desorption process

◦Partition: stationary phase is liquid supported on inert solid. Polar stationary phase and non polar mobile phase – retention of polar compounds and elution of non polar compounds

◦ Ion exchange: stationary phase is an ion exchange resin. Separation mechanism based on ion exchange equilibria .

◦Size exclusion: molecules separated according to size by ability to penetrate a seivelike structure stationary phase. (small molecules longer elution time)

Elution chromatography on columns.

Elution chromatography on columns…Cont

◦ Elution involves washing a species through a column by continuous addition of fresh solvent.

◦ A small volume of sample is placed at the top of the column which is filled with the chromatographic particles (stationary phase) and solvent.

◦ The sample components will then distribute themselves between the two phases (The individual components interact with the stationary phase to different degree)

◦ Introduction of additional mobile-phase forces the solvent containing a part of the sample down the column where further partition between the mobile phase and fresh portions of the stationary phase occurs.

◦ Simultaneously, partitioning between fresh solvent and the stationary phase takes place at the original sample.

◦ Continued addition of solvent carry solute molecules down the column in a continuous series of transfer between the mobile and stationary phases.

Elution chromatography on columns…Cont

◦ Solute movement can only occur in the mobile phase, at which a solute zone migrates down the column depends upon the fraction of time it spends in that phase.

◦ This fraction is small for solutes that are strongly retained by the stationary phase

◦ It is large where retention in the mobile phase is more likely.◦ The resulting difference in rates causes the components in a

mixture to separate into bands/zones located along the column.

◦ Isolation of the separated species is then accomplished by passing a sufficient quantity of mobile phase through the column to cause the individual zones to pass out to the end where they can be detected or collected.

◦ Detector that responds to solute concentration is placed at the end of column (signal is plotted against time) - a series of peaks obtained called chromatogram.

◦ Retention time of sample is obtained. Must be compared with standard.

◦ Chromatogram useful for qualitative and quantitative analysis

Thin Layer Chromatography

Thin Layer Chromatography-Cont..Planar chromatographysimple, rapid versatile sensitive, inexpensive analytical

technique for the separation of substance.The mobile phase is a liquid The stationary phase consists of a thin layer of absorbent

material, which is usually a silica gel, aluminium oxide or cellulose immobilized onto a flat inert carrier sheet.

The developing solvent or the mobile phase is the transport medium for the solutes to be separated as it is migrated through the stationary phase by capillary forces

The movement of substances during TLC is the result of two opposing forces, the driving force of the mobile phase and the resistive or retarding action of the sorbent stationary phase

The driving force tends to move the substances from the origin in the direction of the mobile phase flow

Thin Layer Chromatography-Cont..

The resistive force impedes the movement of the substances by dragging them out of the flowing phase back onto the sorbent.

Each molecule alternates between a sorbed and unsorbed condition, following a stop- and-go path through the sorbent

At the end of development, each substance has migrated a certain mean distance

Substance that move slower are more attracted to the absorbent material, whereas those that move quickly spend a smaller fraction of their time in the layer because of less affinity to it.

GAS CHROMATOGRAPHY

• Separates gaseous substances based on adsorption on or partitioning in a stationary phase from a gas phase• Mobile phase is a gas (Ar, He, N2). • Sample is converted to the vapor state by injection into a heated port and the eluent is a gas.• Stationary phase is a nonvolatile liquid supported on a capillary wall or inert solid particles.

LIQUID CHROMATOGRAPHY(HPLC)

HPLC-Schematic Diagram

HPLC…Cont◦ The system is made up of a high pressure solvent

pump, an injector, a column, a detector and a data recorder

◦ elevated pressure is applied to force a liquid or a liquid mixture through a packed bed of the stationary phase, which is the column, to separate the sample mixture

◦ The pressurized mobile phase passes through the injector and into the column where it equilibrates with the stationary phase

◦ Under the optimal conditions the components to be separated passes through the stationary phase at different velocities and leave the column at different times.

◦ The components are then registered by a detector.◦ This information is passed on to the data evaluation

unit, recorder, and the output is a chromatogram.

HPLC…Cont◦ The number of peaks is equal to the number

of separated components in the sample and the area is proportional to the amount of component

◦ The key to changing the separation is to change the difference in polarity between the column packing and the mobile phase.

◦ Increasing the difference in polarities between column and mobile phase makes compounds stick tighter and come off later. The longer a mixture stays on the column, the better the chance for a separation to occur.

HPLC…Cont

Normal-Phase Chromatography: Highly polar stationary phases such as triethylene glycol or water,a relatively non-polar solvent such as hexane or i-propyl ether served as the mobile phase.

The least polar component is eluted first; increasing the polarity of the mobile phase then decrease the elution time

HPLC…Cont

Reverse-Phase Chromatography: the stationary phase is non-polar such as hydrocarbons and the mobile phase is a relatively polar solvent such as water, methanol, acetonitrile or tetrahydrofuran

The most polar component elutes first, and increasing the mobile-phase polarity, increases the elution time (refer fig 28-14, pg 829)

HPLC…Cont

Case Study HPLC- Determination Arsenic in Urine Sample

Case Study HPLC- Determination Arsenic in Urine Sample

While arsenic is often considered to be the synonym for toxin, arsenic toxicity depends strongly on the species being present. Humans are exposed primarily through ingestion via diet (drinking water, seafood) or inhalation and workplace exposure. Arsenic is used in the manufacture of glass, pigments, medicinals, pesticides, wood preservation and semiconductor products. Once ingested, arsenic gets metabolised to a certain degree depending on speciation end level of exposure and is predominantly excreted in the urine

In order to buffer the changing matrix between different urine samples but also avoid excessive total salt concentrations that would compromise long term stability, a mixture of sodium acetate and sodium nitrate was used as the mobile phase. For enhancing the sensitivity, 1% of ethanol was added as a modifier. 

All five main arsenic species present in human urine (As(III), As(V), AB, MMNA, DMA) were resolved from each other and from chloride within a separation time of 12 min. Detection limits were excellent and ranged from 0.05 to 0.1 µg/l depending on species. Reproducibility of peak area was better than 3% at 10 µg/L and reproducibility of the retention time was better than 0.6%. The arsenic species determined in the CRM NIES No. 18 agreeed well with the certified values vor DMA and AB.

Sample: Analysis Condition