Course CHEM 340 - Chromatography - Chromatographic Methods o Gas Chromatography (GC) o High performance Liquid Chromatography (HPLC) Terms Stationary phase A fixed place either in a column or on a planer surface. Mobile phase A phase that moves over or through the stationary phase carrying with it the analyte mixture. Elution A process in which analytes are washed through a stationary phase by the mobile phase. Eluent A solvent used to carry components in the mixture through the stationary phase. Analyte the substance to be separated Chromatogram the output from the chromatograph Chromatography is the process of separating components which are distributed between a stationary phase and a flowing mobile phase according to the rate at which they are transported through the stationary phase. Separation is based on partition between two phases. Chromatography allows separation, identification and determination of components in complex mixtures. These measurements depend on the availability of the pure compounds as standards, unless mass spectrometry is used. There are several related but different techniques which enable complex mixtures to be separated and the components to be quantified. Typically, a sample is placed on a liquid or stationary phase and a liquid or gaseous mobile phase is passed over it in a process called elution. Solutes whose distribution ratio between the stationary phases differs will be eluted at different rates and this leads to their separation over time and distance. Separation typically occurs by partition or by adsorption. 1
- Partition Chromatography - the mobile and stationary phases are liquids. - Adsorption Chromatography- The mobile phase is a liquid and the stationary phase is a solid. The stationary phase may be in a column/tube or on a plane; therefore chromatographic techniques can be generally divided into column chromatography and planer chromatography. Furthermore they can be classified based on the mobile and stationary phases used as explained below: Planer Chromatography - Paper chromatography (PC) - Thin layer chromatography (TLC) Column Chromatography - Liquid chromatography (LC) -The mobile phase is pumped through a column packed with stationary phase usually at a high pressure. Variants include: o High performance liquid chromatography(hplc)- normal phase or reverse phase o ion-chromatography (ion exchange) - Gas Chromatography (GC) The mobile phase is a pressurized gas o Gas-solid chromatography (Gas MP, Solid SP) - The stationary phase may be small particles of a suitable solid e.g. silica o Gas-liquid chromatography (Gas MP, liquid SP) - The stationary phase is made up of small particles coated with a non-volatile liquid. o Variants include: packed column GC capillary chromatography supercritical fluid chromatography - Capillary chromatography o Small bore capillaries are used often with stationary phase coated on the inside of the wall 2
Other specific Chromatography modes - Electrochromatography ( this is a hybrid technique) - Size-exclusion Chromatography (SEC) - Also called gel-permeation chromatography (GPC), the mobile phase is a solvent and the stationary phase is a packing of porous particles. (can be planar or on column) Paper Chromatography The stationary phase is made of highly purified cellulose but paper treated with silicone or paraffin oil can be used in reverse phase paper chromatography in which a mobile phase is a polar solvent. There are also commercially available papers that contain adsorbents or an ion exchange resin which can be used in adsorption paper chromatography and ion exchange paper chromatography respectively. The mobile phase can be an organic solvent (in normal phase) or a polar solvent in reverse phase. Thin layer Chromatography Thin layer chromatography is performed on a glass plate that is coated with a thin layer of finely divided particles of the stationary phase. The stationary phase can be polar, non-polar or even an ion exchange resin so as to result in specialized applications. A thin-layer plate is prepared by spreading a suspension of a finely ground solid onto a clean surface of glass plate, plastic plate or microscope slide. A binder can be incorporated in the suspension to enhance the binding of the particles onto the plate and to one another. The plate is then left to stand or heated to allow adhesion of the stationary phase onto the plate. A sample is placed near the edge of the plate and the position is marked. After the sample solvent has evaporated, one end of the plate is immersed in the developing solvent/mobile phase (care should be taken to avoid direct contact between the sample and the developer). After the mobile phase has traversed 3
about two thirds of the plate, the plate is removed from the solvent container and dried. The analytes can be located by spraying the plate with reagents which colour the components for example Iodine, sulphuric acid or ninhydrin. Alternatively, a fluorescent material can be incorporated in the stationary phase and after development, the plate is examined under UV light. The plate will fluoresce except at point where the sample components are positioned. Separation of Components in Chromatography The compounds to be separated must be able to distribute themselves between two phases. Therefore they must be volatilized into the gas phase or soluble in the liquid mobile phases and absorbed onto the solid or liquid phase. This is because chromatographic separations are based on the differences in the extent to which analytes are distributed between the mobile and stationary phases. For a solute species A, the equilibrium involved is described by the equation: Component separation is based on the rate of migration of the solute through a stationary phase and this is determined by its distribution ratio. From the equation, large values of K lead to slow solute migration while small values of K lead to rapid solute migration. Solutes are eluted in order of increasing distribution ratio (K). The larger the difference in the distribution ratios of the solutes in the mixture, the more easily they can be separated. 4
Gas Chromatography (GC) The mobile phase in gas chromatography is generally an inert gas. The stationary phase is generally an adsorbent or liquid distributed over the surface of a porous, inert support. In GC a sample is volatilized (vaporized) and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Compounds that cannot be volatilized cannot be separated by GC they can be separated by liquid chromatography or be derivatised to a volatile species. Basic Scheme of a GC Components of a gas Chromatograph Carrier gas The carrier gas must be chemically inert e.g nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependent upon the type of detector which is used. 5
Sample injection port The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column which is maintained at about 50 C (higher than the boiling point of the least volatile component of the sample). For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. Columns There are two general types of column, packed and open tubular. Open tubular columns can be: 1. Wall-coated open tubular (WCOT) 2. Support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns. 6
Column temperature Column temperature can affect how well the analytes are separated. The optimum column temperature is dependent upon the boiling point of the sample. If a sample has a wide boiling range, then temperature programming can be useful. Temperature Programming The Column temperature is increased either continuously or in steps as the separation proceeds. It is achieved by continuously raising the column temperature, usually as a linear function of time, during the elution process. The retention time of a solute is proportional to the distribution coefficient which, in turn, increases as the negative exponent of the standard energy of distribution divided by the product of the gas constant and the absolute temperature Detectors Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors (The signal from a concentration dependant detector is related to the concentration of 7
solute in the detector) mass flow dependant detectors (the signal is related to the rate at which solute molecules enter the detector) The Flame Ionization Detector The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample. 8
Summary of GC Detectors Detector Type Support gases Selectivity Detectio n range Flame ionization Mass flow Hydrogen Most organic cpds. 100 pg (FID) and air Thermal Concentrati Reference Universal 1 ng conductivity (TCD) on Electron capture Concentrati Make-up Halides, nitrates, nitriles, 50 fg (ECD) on peroxides, anhydrides, organometallics Nitrogenphosphorus Mass flow Hydrogen Nitrogen, phosphorus 10 pg and air Flame Mass flow Hydrogen Sulphur, phosphorus, tin, 100 pg photometric (FPD) and air or boron, arsenic, oxygen germanium, selenium, chromium Photo-ionization Concentrati Make-up Aliphatics, aromatics, 2 pg (PID) on ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics Choice of a detector is based on Sensitivity Selectivity for different classes of compounds Linearity, the bigger the linear range the better Cost Note - Multiple detectors can be used, effluent is split at the end of column 9
Liquid Chromatography (LC) The most common type of column liquid chromatography is high performance liquid chromatography (HPLC), where a pump is used to force the mobile phase a packed column. The sample is injected at the head of the column and a detector at the end of the column monitors the separated analyte components which are recorded (on a PC). HPLC is used to separate nonvolatile or thermally unstable compounds (e.g Biological compounds, pharmaceuticals, low- or non-volatile environmental compounds. e.g. PCB, DDT). HPLC separations result from the mechanism of interaction of the analytes with the stationary phase and the relative polarities of the stationary and mobile phases. Commonly used stationary phases may be small rigid porous particles with a high surface area but monoliths are also being used. The main parameters to consider are: - Particle size(1.8 10 µm)- Small sizes better however the lead to high back up pressure - Particle size distribution as narrow as possible - Surface area (50 250 m 2+ /g) the bigger the better - Bonding phase density the number of adsorption sites per surface unit In addition the polarity of the eluent/mobile phase plays a big role in the separation. Elution can be isocratic (eluent composition is constant, polarity of eluent is constant) or gradient (eluent composition is changed during the analysis in effect changing the polarity of the eluent as analysis goes on). The choice of mobile phase is based on the polarity of the solvent and the following parameters should also be considered - Purity - Stability (Chemical and thermal) - Viscosity - Compatibility (with the stationary phase and the detector 10
Basic/ Typical HPLC components: Liquid Mobile Phase => Pump => Injection valve => Separation Column => Detector An integrator records the detector response. Sample Injection - Liquid samples or solutions are introduced into a flowing mobile phase at the top of the column through a constant or variable volume loop and valve injector which is loaded with a syringe (similar to GC) Small samples (0.1-100 ml) can be introduced without depressurization Microsyringe/septum system can be used only at pressure <1500 psi In the case of HPLC, the pressure within the column makes direct injection into the mobilephase difficult. Instead a specialist injection system, such as a Rheodyne system that incorporates a sample loop is used. 11
The solvent is usually degassed, filtered and blended (if more than one is used) before it is delivered to the top of the column under pressure by a constant flow pump. 12
HPLC Columns They are straight lengths of stainless steel, 10-30 cm long, 4-10 mm internal diameter. They are tightly packed with a micro particulate stationary phase (1-10 mm particle size - 40,000-60,000 plates/m). The column packings are chemically modified silicas (e.g octadecyl silica, aminopropyl silica, cyanopropyl silica), unmodified silica or polymeric resins or gels. A mixture is separated by differential migration through the column. Modes of HPLC - are defined by the nature of the stationary phase, the mechanism of interaction with solutes and the relative polarities of the stationary and mobile phases Figure taken from D.L Saunders Chromatography, third edition, E heftmann, p 81, Newyork: Van Nostrand Reinhold, 1975. 13
Adsorption Chromatography Separation is normal phase commonly with a silica gel stationary phase and a mobile phase of a non-polar solvent blended with addition of a more polar solvent to adjust the overall polarity (e.g hexane + dichloromethane). Solutes are retained by surface adsorption [ they compete with solvent molecules for active silanol sites (SiOH)] and are eluted in order of increasing polarity. Partition Chromatography This is the most common mode of HPLC. Chemically modified silicas or bonded phases are used with the most common being non polar hydrocarbons. The solute sorption mechanism is a modified partition because although the bonded hydrocarbons are not true liquids organic solvent molecules from the mobile phase from a liquid layer on the surface. Most separations are reverse phase although the normal phase can also be used. Methanol or acetonitrile with water or an aqueous buffer is the most common blend of mobile phase for the reverse phase and the stationary phase is octadecyl silica (C18 or ODS). This combination can be used in separation of moderately polar to polar solutes. Ion exchange Chromatography (IEC) This mode is applied in the separation of mixtures of ionic solutes. The stationary phases are either micro particulate ion-exchange resins or bonded phase silicas. Both types have either sulfonic acid cation-exchange sites (-SO 3 - H + ) or quaternary ammonium anion exchange site (-N + R 3 OH - ) incorporated into their structures Mechanism of ion exchange Ions held on a porous insoluble solid are exchanged for ions in a solution which is brought into contact with the solid. Synthetic ion exchange resins have been widely used in water softening, deionization, solution purification and ion separation. 14
Ion exchange resins Cation-exchange resins contain acidic groups while anion exchange resins have basic groups. Illustration of a cation exchange xrso - 3 H + + M x+ = (RSO - 3 ) x M x+ + xh + Solid soln solid soln Where M x+ represents a cation and R represents the part of a resin molecule that contains one sulphonic acid group Illustration of anion exchanger xrn(ch 3 ) + 3 OH - + A x- = [RN(CH 3 ) + 3 ]A x- + xoh - Solid soln solid soln Where A x- represents the anion Example. When a dilute solution of calcium ions is passed through a column packed with a sulphonic acid resin the following equilibrium is established Ca 2+ (aq) + 2H + (res) = Ca 2+ (res) + 2H + (aq) The equilibrium constant is given by [ ] [ ] [ ] [ ] Ion exchange separations are usually performed under conditions in which one ion predominates in both phases. e.g in the removal of calcium ions from a dilute solution, the calcium ion concentration will be much smaller than that of the hydrogen ion concentration in both the aqueous and in the resin phases [Ca 2++ ] res << [H + ] res and [Ca 2++ ] aq << [H + ] aq Therefore, the hydrogen ion concentration is essentially constant in both phases and the equation above can be written as: [ ] [ ] 15
Where K is the distribution constant (similar to the one obtained from extraction equilibrium) Generally, when K is large, there is a strong tendency for the resin to retain the ion. The opposite is true when K is small. Differences in the values of K are related to the size of the hydrated ion and polyvalent ions are more strongly retained than singly charged species. For a typical sulphonated cation exchange resin, the values of K for univalent ions decreases in the order Ag + > Cs + > Rb + > K + > NH 4 + > Na + > H + > Li +. For divalent ions, the order is Ba 2+ > Pb 2+ > Sr 2+ > Ca 2+ > Ni 2+ > Cd 2+ > Cu 2+ > Co 2+ > Zn 2+ > Mg 2+ > UO 2 2+. Applications of ion exchange 1. Elimination of interfering ions in an analysis 2. Concentration of ions from a dilute solution 3. Chromatographic separation of inorganic and organic ion species 4. Water softeners (read) Size exclusion Chromatography (SEC) This separation method is based on sieving rather than partitioning. The column is filled with a porous stationary phase (pore size is controlled) and the sample is separated based on molecular size. Smaller size molecules penetrate through the pores and are therefore retained longer while larger molecules are excluded and therefore washed down the column faster (first to be eluted). SEC is mainly used in separation of large molecules such as proteins and polymers. Chromatographic Performance The time taken for a solute to pass through a column of separation is referred to as retention time (t R ). The figure below show a simple chromatogram in which the first peak show the component which is not retained by the stationary phase. The time taken for this peak to appear is called dead or void time. It is the time taken for the mobile phase to be eluted. 16
Migration rate (v in cm/s) of the solute can be obtained from: Where L is the length of the column packing Similarly the average linear velocity, u, of the mobile-phase molecules is To relate the migration rate of a solute to its distribution constant, the rate is expressed as a fraction of the velocity of the mobile phase: Chromatographic performance can be assed using the following factors Partition/Distribution coefficient (K) An equilibrium expression that describes the distribution of an analyte between two different phases (stationary and mobile). Retention/capacity factor (k) It is the amount of time a solute spends in the stationary phase relative to the time it spends in the mobile phase. This is used to compare migration rates of analytes on columns. The retention factor for analyte A is defined as; k A = t R - t M / t M t R and t M are easily obtained from a chromatogram. 17
Optimally, k should have a value between 1 and 10; <1 results in poor separation (analyte comes off with mobile phase) and >10 results in a lot of time for peaks to elute. This factor is independent of flow rate it is dependent on physical interactions. Selectivity factor (α) It describes the separation of two species (A and B) on the column. It is the ratio of the retention factors i.e. α = k B / k A where, species A elutes faster than species B. The selectivity factor is always greater than one. Resolution (R s ) Describes the separation among solutes i.e how far apart two bands are relative to their widths. The selectivity factor, α, describes the separation between analytes but it does not take into account peak widths. Measurement of the resolution explains how well species have been separated. The resolution of two species, A and B, is defined as Baseline resolution is achieved when R = 1.5 NOTE: Separations can be improved by - By controlling the capacity factor, k, e.g by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography). - Controlling the selectivity factor, α, e.g by 1. Changing mobile phase composition 2. Changing column temperature 3. Changing composition of stationary phase 18
4. Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase). Height equivalent to the theoretical plate (HETP) This relates to the number of plates required for an efficient separation and it is derived from the theoretical plate model of chromatography. The plate model supposes that the chromatographic column contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. [It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better)]. If the length of the column is L, then the HETP is HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution; 19
Where w 1/2 is the peak width at half-height. As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture. The Rate Theory of Chromatography Rate Theory examines the various forces that produce band broadening and affect precision. These include -The flow rate of the mobile phase -The different paths through which solute molecules travel between particles of the stationary phase A more realistic description of the processes is based on the time taken for the solute to equilibrate between the stationary and mobile phase (which is very fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. These mechanisms contribute to band broadening and can be accounted for by the Van Deemter equation for plate height: HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening. A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte 20
spends less time on the column, which decreases the effects of longitudinal diffusion. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes. Van Deemter plots A plot of plate height vs. average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate. The efficiency of a chromatographic column is affected by the amount of band broadening which occurs when a compound passes through the column. An ideal elution peak should look like the Gaussian/normal curve. However some peaks exhibit Tailing or fronting. 21
Illustration Tailing and fronting is due to the variation of distribution constant with concentration. Fronting can also result when a large amount of sample is injected into the column is too large. These distortions in peak shapes lead to poor separations and non-reproducible retention times. The efficiency of a column can be described based on plate height (H) or on the number of theoretical plates (N) Where L is the length (in cm) of the column packing 2 is the variance obtained from the Gaussian curve References Skoog, West, Holler & Crouch (2004), Fundamentals of Analytical Chemistry, 8 th Ed, Thomson Brookes Publishers Harris D.C.(2005), Exploring Chemical Analysis, New York 22