Assignment Institute of Advance Research Studies in Chemical Sciences (IARSCS), Hi-Tech Research Laboratories, University of Sindh, Jamshoro, Pakistan.

Introduction, classification and types of Chromatographic Techniques

Assigned By : Dr Abdul jabbar laghari Submitted By: s.umair jarwar

Introduction to chromatography, its types and classification Chromatography Chromatography is a set of techniques in which separation of chemical substances takes place quantitatively as well as qualitatively. Terminology used in Chromatography Mobile Phase: In chromatography the substance which is introduced with or along with the sample and causes elution of the contents of the sample. It may be liquid or gas. Stationary phase: Stationary phase of the chromatographic system refers to that part which is present before the introduction of sample or solute in the column ( as in column chromatography) or on a solid support( as in paper or similar chromatography). It may be liquid or solid. Eluent: The substance which separates the components of the mixture in chromatographic technique. Eluent is that part which brings separation when the solution is passed either from the column or from the solid support. Eluate: The substance which is separated as a individual component of the mixture is called eluate.

Classification of Chromatographic Techniques

Chromatographic methods can be differentiated based on the physical means of bringing the stationary and mobile phases into contact it means the basis of classification is how the stationary base and mobile phase come into contact. Column Chromatography - the stationary phase is held in a narrow tube through which the mobile phase is forced either by pressure or by gravity. Examples include: Simple column chromatography High pressure liquid chromatography (HPLC) Gas chromatography (GC).

Planar Chromatography - the stationary phase is supported on a flat plate or in the fibres of a paper. Here the mobile phase moves through the stationary phase by capillary action or by gravity. Examples include: Paper chromatography Thin layer chromatography (TLC).

Column chromatography can be further differentiatedbased on the types of stationary and mobile phases and the kinds of equilibria involved in solute transfer between the phases. There are two broad categories in this classification scheme:

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Liquid chromatography (for instance simple column or HPLC) Gas Chromatography (GC).

On the basis of polarity of phases classification can be: i. ii. Normal phase Chromatography Reversed Phase Chromatography

On the basis of experimental parameter variation over separation period classification may be: i. ii. iii. iv. Isothermal in which Temperature is kept constant Temperature Programming in which column temperature changes systematically Isocratic in which solvent composition is held constant Solvent Programmed in which solvent composition and concentration changes systematically.

Important types of Chromatographic Techniques Following are some important types of Chromatographic separation techniques. They are defined thoroughly by explaining their general principle, application, and a brief outline of their instrumentation for a complete understanding. Following are some commonly utilized types of techniques: i. ii. iii. iv. Gas Chromatography High Pressure Liquid Chromatography Supercritical fluid Chromatography Gel Exclusion Chromatography

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GAS CHROMATOGRAPHY INTRODUCTION Gas chromatography is commonly used separation technique. Gas chromatography involves mobile carrier gas It has liquid or polymeric stationary phase

NOTABLE POINTS WHICH DIFFERENTIATE GAS CHROMATOGRAPHY FROM OTHER TYPES OF CHROMATOGRAPHY In gas chromatography the stationary phase is liquid In gas chromatography the column is present in an oven where temperature programming takes place The concentration of a compound in the gas phase is solely a function of vapor pressure of the gas.

PRINCIPLE OF GAS CHROMATOGRAPHY ANALYSIS Gas chromatographic system utilized narrow column in which sample is introduced alongwith the carrier gas. Sample passes in the gas stream at a different rate depending upon the physical and chemical properties and the interaction with the stationary phase. When chemical are eluted out they are recorded electronically. The function of the stationary phase is to elute the different components of the sample at different times i.e.at different retention times. The function of the mobile phase is that it carries the sample substance to interact with the stationary phase already present in the column Column length, temperature, and the type of carrier gas can be used as parameters for different retention times and separation.

TYPES OF SAMPLES SEPARATED IN GAS CHROMATOGRAPHY AND ITS APPLICATIONS All types of volatile mixtures

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Air samples Different pharmaceutical samples Cosmetics and environmental toxins

INSTRUMENTATION OF GAS CHROMATOGRAPHY Sample Injection In modern GC units heated samples are introduced via microsyringe A calibrated microsyringe is used to deliver a sample volume in the range of a few microliters through a rubber septum and into the vaporization chamber. Most separations require only a small fraction of the initial sample volume and a sample splitter is used to direct excess sample to waste. Commercial gas chromatographs often allow for both split and splitless injections when alternating between packed columns and capillary columns.

Carrier Gas Carrier gas must be dry, free of oxygen and chemically inert mobile-phase employed in gas chromatography. Helium is most commonly used because it is safer than, but comprable to hydrogen in efficiency, has a larger range of flow rates and is compatable with many detectors. Nitrogen, argon, and hydrogen are also used depending upon the desired performance and the detector being used. Both hydrogen and helium, which are commonly used on most traditional detectors such as Flame Ionization(FID), thermal conductivity (TCD) and Electron capture (ECD), provide a shorter analysis time and lower elution temperatures of the sample due to higher flow rates and low molecular weight.

Column Oven The thermostatted oven serves to control the temperature of the column within a few tenths of a degree to conduct precise work. The oven can be operated in two manners: isothermal programming or temperature programming. In the temperature programming method, the column temperature is either increased continuously or in steps as the separation progresses. This method is well suited to separating a mixture with a broad boiling point range.

Open Tubular and Packed Columns

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Two types of columns can be used in GC. (i) Open Tubular also known as Capillary Column; and (ii) Packed Column Open Tubular column are more efficient and widely used Packed column provide less efficiency Thin layer of stationary phase is coated on open tubular column

Detection Systems used in Gas Chormatography Following detectors can be used in Gas Chromatography: Flame Ionization detectors (FID) used for Hydrocarbon samples and have detection limit upto 1pg/s Thermal Conductivity Detectors ( TCD) used for all range of Chemical substance and therefore it is called as Universal Detector of GC system. Detection limit of this type of detector is up to 500pg/s. Electron Capture Detector (ECD) are used for Halogenated Hydrocarbons and they have a detection limit up to 5fg/s. Photoionization Detectors are used for gaseous and vapor compounds. They have 0.002 to 0.02microgram/L detection limit. Mass spectrometers can be hyphenated with a GC system and it can also be used for higher degree of separation, identification and characterization of compounds.

EQUATION USED IN GAS CHROMATOGRAPHY Height equivalent to a theoretical plate (HETP) use to calculate the flow rate by usingthe total number of theoretical plates (N) and column length (L). Some application, HETP concepts is used in industrial practice to convert number of theoretical plates to packing height. HETP can be calculate with the Van Deemter equation, which is given by

HETP=A+B+Cv

(1)

Where A and B and C are constants and v is the linear velocity (carrier flow rate). A is the "Eddy-Diffusion" term and causes the broadering of the solute band. B is the "Longitudinal diffusion" term whereby the concentration of the analyte, in which diffuses out from the center to the edges.This causes the broadering of the analyte band. C is the "Resistance to Mass Transfer " term and causes the band of the analyte broader.

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ( HPLC)

Chromatographic Theory
Affinities for Mobile and Stationary Phases

All chromatographic separations, including HPLC operate under the same basic principle; every compound interacts with other chemical species in a characteristic manner. All chemical reactions have a characteristic equilibrium constant. For the reaction

Aaq +Bs ABs

There is a chemical equilibrium constant Keq that dictates what percentage of compound A will be in solution and what percentage will be bound to the stationary compound B. During a chromatographic separation, there is similar relationship between compound A and the solvent, or mobile phase, C. This will yield an overall equilibrium equation which dictates the quantity of A that will be associated with the stationary phase and the quantity of A that will be associated with the mobile phase.

Amobile Astationary

The equilibrium between the mobile phase and stationary phase is given by the constant Kc.

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Kc =(aA)S(aA)McScM

Where Kc, the distribution constant, is the ratio of the activity of compound A in the stationary phase and activity of compound A in the mobile phase. In most separations, which contain low concentrations of the species to be separated, the activity of A in each is approximately equal to the concentration of A in that state. The distribution constant indicates the amount of time that compound A spends adsorbed to the stationary phase as the opposed to the amount of time A spends solvated by the mobile phase.

Solvent

The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. The solvent is passed through a very narrow bore column, any contaminants could at worst plug the column, or at the very least add variability to the retention times during repeated different trials. Therefore HPLC solvent must be kept free of dissolved gases, which could come out of solution mid-separation, and particulates.

Column In the HPLC column, the components of the sample separate based on their differing interactions with the column packing. If a species interacts more strongly with the stationary phase in the column, it will spend more time adsorbed to the column's adsorbent and will therefore have a greater retention time. Columns can be packed with solids such as silica or alumina; these columns are called homogeneous columns. If stationary phase in the column is a liquid, the column is deemed a bonded column. Bonded columns contain a liquid stationary phase bonded to a sold support, which is again usually silica or alumina. The value of the constant C described in the van Deemter equation is proportional, in HPLC, to the diameter of the particles that constitute the column's packing material.

Pump

The HPLC pump drives the solvent and sample through the column. To reduce variation in the elution, the pump must maintain a constant, pulse free, flow rate; this is achieved with multi-piston pumps. The presence of two pistons allows the flow rate to be controlled by one piston as the other recharges. A syringe pump can be used for even greater control of flow rate; however, the syringe pump is unable to produce as much pressure as a piston pump, so it cannot be used in all HPLC applications. Detector The HPLC detector, located at the end of the column, must register the presence of various components of the sample, but must not detect the solvent.

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For that reason there is no universal detector that works for all separations. A common HPLC detector is a UV absorption detector, as most medium to large molecules absorb UV radiation. Detectors that measure fluorescence and refractive index are also used for special applications. A relatively new development is the combination of an HPLC separation with an NMR detector. This allows the pure components of the sample to be identified and quantified by nuclear magnetic resonance after having been separated by HPLC, in one integrated process.

Technique
Normal Phase vs. Reverse Phase If the stationary phase is more polar than the mobile phase, the separation is deemed normal phase. If the stationary phase is less polar than the mobile phase, the separation is reverse phase. In reverse phase HPLC the retention time of a compound increases with decreasing polarity of the particular species. The key to an effective and efficient separation is to determine the appropriate ratio between polar and non-polar components in the mobile phase. The goal is for all the compounds to elute in as short a time as possible, while still allowing for the resolution of individual peaks. Typical columns for normal phase separation are packed with alumina or silica. Alkyl, aliphatic or phenyl bonded phases are typically used for reverse phase separation.

Gradient Elution vs. Isocratic Elution If the composition of the mobile phase remains constant throughout the HPLC separation, the separation is deemed an isocratic elution. Often the only way to elute all of the compounds in the sample in a reasonable amount of time, while still maintaining peak resolution, is to change the ratio of polar to non-polar compounds in the mobile phase during the sample run. Known as gradient chromatography, For a reverse phase gradient, the solvent starts out relatively polar and slowly becomes more non-polar. The gradient elution offers the most complete separation of the peaks, without taking an inordinate amount of time. A sample containing compounds of a wide range of polarities can be separated by a gradient elution in a shorter time period without a loss of resolution in the earlier peaks or excessive broadening of later peaks.

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Gradient elution requires more complex and expensive equipment and it is more difficult to maintain a constant flow rate while there are constant changes in mobile phase composition. Gradient elution, especially at high speeds, brings out the limitations of lower quality experimental apparatus, making the results obtained less reproducible in equipment already prone to variation. If the flow rate or mobile phase composition fluctuates, the results will not be reproducible.

Applications
HPLC can be used in both qualitative and quantitative applications, that is for both compound identification and quantification. Normal phase HPLC is only rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for only a few separation types; it cannot separate inorganic ions (they can be separated by ion exchange chromatography). It cannot separate polysaccharides (they are too hydrophilic for any solid phase adsorption to occur), nor polynucleotides (they adsorb irreversibly to the reverse phase packing). Lastly, incredibly hydrophobic compounds cannot be separated effectively by RPLC (there is little selectivity). RPLC is used for the separation of almost all other compound varieties. RPLC can be used to effectively separate similar simple and aromatic hydrocarbons, even those that differ only by a single methylene group. RPLC effectively separates simple amines, sugars, lipids, and even pharmaceutically active compounds. RPLC is also used in the separation of amino acids, peptides, and proteins. Finally RPLC is used to separate molecules of biological origin. The determination of caffeine content in coffee products is routinely done by RPLC in commercial applications in order to guarantee purity and quality of ground coffee. HPLC is a useful addition to an analytical arsenal, especially for the separation of a sample before further analysis.

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Gel permeation chromatography

Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates analytes on the basis of size. The technique is often used for the analysis of polymers.

Gel Permeation Chromatography Theory GPC separates based on the size or hydrodynamic volume (radius of gyration) of the analytes. This differs from other separation techniques which depend upon chemical or physical interactions to separate analytes.[3] Separation occurs via the use of porous beads packed in a column The smaller analytes can enter the pores more easily and therefore spend more time in these pores, increasing their retention time. Larger analytes spend little if any time in the pores and are eluted quickly. All columns have a range of molecular weights that can be separated.

Applications GPC is often used to determine the relative molecular weight of polymer samples as well as the distribution of molecular weights. GPC truly measures is the molecular volume and shape function as defined by the intrinsic viscosity. If comparable standards are used, this relative data can be used to determine molecular weights within 5% accuracy. Polystyrene standards with PDI of less than 1.2 are typically used to calibrate the GPC.

Instrumentation Gel permeation chromatography is conducted almost exclusively in chromatography columns. The experimental design is not much different from other techniques of liquid chromatography. Samples are dissolved in an appropriate solvent, in the case of GPC these tend to be organic solvents and after filtering the solution it is injected onto a column. A Waters GPC instrument is shown to the left. The separation of multi-component mixture takes place in the column.

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The constant supply of fresh eluent to the column is accomplished by the use of a pump. Since most analytes are not visible to the naked eye a detector is needed. Multiple detectors are used to gain additional information about the polymer sample. The availability of a detector makes the fractionation convenient and accurate.

Gels Gels are used as stationary phase for GPC. The pore size of a gel must be carefully controlled in order to be able to apply the gel to a given separation. Other desirable properties of the gel forming agent are the absence of ionizing groups and, in a given solvent, low affinity for the substances to be separated. Commercial gels like Sephadex, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel are often used based on different separation requirements.

Mobile Phase The mobile phase should be a good solvent for the polymer, should permit high detector response from the polymer and should wet the packing surface. The most common eluents in for polymers that dissolve at room temperature GPC are tetrahydrofuran (THF), o-dichlorobenzene and trichlorobenzene at 130150 C for crystalline polyalkynes and m-cresol and o-chlorophenol at 90 C for crystalline condensation polymers such as polyamides and polyesters.

Detectors In GPC, the concentration by weight of polymer in the eluting solvent may be monitored continuously with a detector. There are many detector types available and they can be divided into two main categories. The first is concentration sensitive detectors which includes UV absorption, differential refractometer (DRI) or refractive index (RI) detectors, infrared (IR) absorption and density detectors. Molecular weight sensitive detectors include low angle light scattering detectors (LALLS), multi angle light scattering (MALLS). The resulting chromatogram is therefore a weight distribution of the polymer as a function of retention volume. The most sensitive detector is the differential UV photometer and the most common detector is the differential refractometer (DRI).

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When characterizing copolymer, it is necessary to have two detectors in series. For accurate determinations of copolymer composition at least two of those detectors should be concentration detectors. The determination of most copolymer compositions is done using UV and RI detectors, although other combinations can be used.

References 1. Principles of Instrumental Analysis By Douglas A. Skoog 2. Quantitative Chemical Analysis by Daniel C. Harris 3. Fundamentals Of Analytical Chemistry by David Harvey 4. Analytical Chemistry by G.D Kristen
5. Determination of vitamin E in biological specimens and foods by HPLC-pretreatment of samples
and extraction of tocopherols by T Ueda, O Igarashi - Journal of micronutrient analysis, 1990

6. Separation of Single-Walled Carbon Nanotubes by 1-Dodecanol-Mediated SizeExclusion Chromatography BS Flavel, MM Kappes, R Krupke, F Hennrich - ACS nano, 2013 ACS Publications

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