INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS

What is chromatography? Chromatography is a powerful separation method that is usually composed of mobile phase and a stationary phase. This method is used to separate and identify the components of complex mixtures. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile phase to varying degrees.

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 that can be analyzed qualitatively and/or quantitatively.

Classification of Chromatographic Methods Can be categorized based on the followings: 1. Based on physical means The way stationary and mobile phase are brought into contact 2. Based on the types of mobile phase Either gas, liquid or supercritical fluid 3. Based on the kinds of equilibria involved in the solute transfer between the phases. Interaction of analyte between stationary and mobile phases

Classification of Chromatographic Methods Column chromatography Planar chromatography Stationary phase is supported on a flat plate or in the interstices of a paper; mobile phase moves through capillary action or gravity E.g. – thin-layer chromatography (TLC) – paper chromatography (PC) Stationary phases is held in narrow tube; mobile phase moves by pressure or gravity E.g. – gas chromatography (GC) – supercritical-fluid chromatography (SFC) * Based upon physical means

Column chromatography can be further differentiated based on the types of mobile phases and the kinds of equilibria involved in solute transfer between the phases Mobile Phase i) Gas Gas Chromatography iii) Supercritical fluid Supercritical-fluid Chromatography ii) Liquid Liquid Chromatography

Types of Chromatography on The Basis of interaction of The Analyte with Stationary Phase Adsorption – for polar non-ionic compounds Ion Exchange – for ionic compounds Anion – analyte is anion; bonded phase has positive charge Cation – analyte is cation; bonded phase has negative charge Partition – based on the relative solubility of analyte in mobile and stationary phases Normal – stationary phase polar, the mobile phase nonpolar Reverse – stationary phase nonpolar, the mobile phase polar Size Exclusion – stationary phase is a porous matrix sieving

Classification of Chromatographic Methods Chromatography Partition Adsorption Ion-exchange Size-exclusion Liquid-liquid Liquid-solid Liquid-solid Liquid-solid Gas-liquid Gas-solid

Partition Chromatography

Partition chromatography Accomplished by selective & continuous transfer of the components of the mixture back & forth between a liquid stationary phase and a liquid mobile phase as the mobile phase liquid passes through the stationary phase liquid Stationary phase: liquid Mobile phase: liquid or gas

e.g. stationary phase – polar Partitioning distribution (by dissolving) of the components between 2 immiscible phases: Relative solubilities of the components in the mobile and stationary phase e.g. stationary phase – polar Polar components will retain longer than the non-polar components. Non-polar components will move quickly through stationary phase & will elute first before the polar components, and vice-versa.

Partition chromatography The stationary phase actually consists of a thin film adsorbed (stuck) on or chemically bonded to the surface of a finely divided solid particles.

Partition chromatography If the mobile phase is gas, the volatility (vapor pressure) and solubility in stationary phase plays an important role.

Adsorption Chromatography

Adsorption (Affinity) Chromatography Components of the mixture selectively adsorb (stick) on the surface of a finely divided solid stationary phase. As mobile phase (gas/liquid) carries the mixture through the stationary phase, the components of the mixture stick to the surface of it with varying degrees of strength & thus separate Stationary phase: solid Mobile phase: gas or liquid

Ion-exchange chromatography

Ion-exchange chromatography Method for separating mixture of ions Sample: aqueous solution of inorganic ions / organic ions Stationary phase – small polymer resin “beads” usually packed in a glass tube These beads have ionic bonding sites on their surfaces which selectively exchange ions with certain mobile phase compositions as the mobile phase penetrates through it.

Ion-exchange chromatography Ions that bond to the charged site on the resin bead are separated from ions that do not repeated changing of the mobile phase composition. The usual procedure is to initially use a mobile phase with all the ions in the mixture bond & then to change the mobile phase in a stepwise fashion so that one kind of ion at a time is removed Done until complete separation achieved

Size-exclusion chromatography

Size-exclusion chromatography Also called gel permeation chromatography Technique for separating dissolved species on the basis of their size Stationary phase: porous polymer resin particles (molecular sieves) The components to be separated enter the pores of these particles & are slowed from progressing through this stationary phase.

Size-exclusion chroamtography Separation depends on the sizes of the pores relative to the sizes of the molecules to be separated Small particles are retarded to a greater extent than large particles (some of which may not enter the pores at all) & separation occurs.

Terminologies in chromatography

Terminologies in chromatography Elution: a process in which species are washed through a chromatographic column by addition of fresh solvent Mobile phase: is one that moves over or through an immobilized phase that is fixed in place in a column or on the surface of flat plate Stationary phase: a solid or liquid that is fixed in place. A mobile phase then passes over or through the stationary phase Retention time: is the time interval btw its injection onto a column and the appearance of its peak at the other end of the column

Migration Rates of Solutes Distribution constant, K Retention time, tR Capacity factor,k’ Selectivity factor, 

Distribution constant, K In chromatography, the distribution equilibrium of analytes between the mobile and stationary phases can often be described quite simple. Let say, we have analyte A. The distribution equilibrium is written as: A mobile  A stationary Therefore, the equilibrium constant K is called distribution constant and is defined as: K = c – Molar concentration cstationary cmobile K is also called partition coefficient or partition ratio

Retention Time, tR Time required for the sample to travel from the injection part through the column to the detector. A typical chromatogram for a two-component mixture. The small peak on the left represents a species that is not retained on the column & so reaches the detector almost immediately after elution is started.

tM - time taken for the unretained species to reach the detector. - sometimes called dead time - Rate of migration of the unretained species is SAME as the average rate of motion of mobile phase molecules. - So, tM can be expressed as the time required for an average molecule of the mobile phase to pass through the column.

Retention Factor (Capacity factor), k’ term used to measure the migration rates of analytes on columns. k’A = KA (VS / VM) [unitless] for analyte A How is k’A related to tR and tM? k’A = tR – tM tM When k’A is  1.0, separation is poor When k’A is > 30, separation is slow When k’A is 2-10, separation is optimum

Selectivity Factor,  is defined as:  = = KB KA k’B A measure of the relative migration rates of species A and B with a stationary phase material in chromatography KB KA distribution constants k’B k’A capacity factors tR(B) – tM tR(A) – tM retention times

tR – tM tM tR(B) – tM tR(A) – tM k’ =  = Response tR tR tM 1 3 6 Retention time , min tR – tM tM tR(B) – tM tR(A) – tM k’ =  =

Column Efficiency Two related terms widely used as quantitative measures of chromatographic column efficiency: i) Plate height, H ii) Number of theoretical plates, N

Efficient column has small plate height The relationship between H and N is: N = The efficiency of chromatographic columns increases as the number of plates becomes greater and plate height become smaller. Column length L H Plate height Number of theoretical plates Efficient column has small plate height

Experimentally, H and N can be approximated from the width of the base of the chromatographic peak. The equation: 2 tR W N = 16 N can be calculated using tR and W To obtain H, the length of the column must be known

Another method for approximating N is to determine W½, the width of the peak at half its maximum height. N = 5.54 tR 2 W½

Resolution, Rs A measure of the separation of two chromatographic peaks. Baseline resolution is achieved when Rs = 1.5 Rs = 2[tR(B) – tR(A)] WA + WB

Effect of Capacity Factor & Selectivity Factor on Resolution Relationship btw the resolution of a column and the capacity factor k’, selectivity factor  and the number of plates N is given by this equation: Rs = √N  - 1 k’ 4  1 + k’ Simplified: Rs = √N

Effect Resolution on Retention Time Relationship btw the resolution of a column and retention time: 2 tR = 16Rs2H  ( 1 + k’)3 u  - 1 (k’)2 Simplified: tR = Rs2

Peak widths (at base) for A & B were 1.11 & 1.21 min respectively. Example Length of column: 30 cm Peak widths (at base) for A & B were 1.11 & 1.21 min respectively. Calculate: i) column resolution, Rs ii) the average number of plates, N iii) the plate height, H iv) length of column to achieve Rs 1.5

17.63 min Response tR 16.40 min tR 1.30 min tM 1 3 6 Retention time , min

i) Rs = 2(17. 63 min – 16. 40 min) (1. 11 min + 1. 21 min) = 1 i) Rs = 2(17.63 min – 16.40 min) (1.11 min + 1.21 min) = 1.06 ii) N = 16 16.40 min 1.11 min = 3.49 x 103 Rs = 2[tR(B) – tR(A)] WA + WB 2 tR W N = 16 2

2 N = 16 17.63 min 1.21 min = 3.40 x 103 H = L / N = 30 cm / 3.44 x 103 = 8.7 x 10-3 cm (Rs)1 √N1 (Rs)2 √N2 Therefore, calculate the N average Nave = 3.44 x 103 =

1.06 = √ 3.44 x 103 1.5 √ N 2 N2 = 6.9 x 103 L = N x H = 6.9 x 103 x 8.7 x 10-3 = 60 cm

BAND BROADENING Band broadening reflects a loss of column efficiency. The slower the rate of mass-transfer processes occuring while a solute migrates through a column, the broader the band at the column exit. Some of the variables that affect mass-transfer rates are controllable and can be exploited to improve separations. Table 26.2 lists the variables that influence the column efficiency. Their effect on column efficiency, as measured by the plate height will be described in the following slides

VARIABLES AFFECTING COLUMN EFFICIENCY

VARIABLES AFFECTING COLUMN EFFICIENCY Mobile phase flow rate Particle size Diameter of column Film thickness

EFFECT OF MOBILE PHASE FLOW RATE ON PLATE HEIGHT From both the plots for LC and GC, we can see that both show a minimum in H at low linear flow rates.

EFFECT OF PARTICLE SIZE ON PLATE HEIGHT Refer to figure 26-11 , page 773 The numbers to the right is the particle diameters The smaller the particle size, the more uniform the column packing, then the more tolerant to the change in mobile-phase velocity.

EFFECT OF DIAMETER OF THE COLUMN ON PLATE HEIGHT For packed column, the most important variables that affect column efficiency is the diameter of the particles that making up the packing. While for open tubular column, the diameter of the column itself is an important variables. Refer to table 26-3, the mobile phase mass- transfer coefficient CM is known to be inversely proportional to the diffusion coefficient of the analyte in the mobile phase DM.

CM is proportional to the square of the particle diameter of the packing material, d1p (packed column). CM is proportional to the square of the column diameter, d2p (open tubular column). As a conclusion, the bigger the column diameter, the smaller the diffusion coefficient DM. therefore, we can say that increase in column diameter will increase the plate height.

EFFECT OF FILM THICKNESS ON PLATE HEIGHT When stationary phase is an immobilized liquid, the mass-transfer coefficient Cs is directly proportional to the square of the thickness of the film on the support particles d1l and inversely proportional to the diffusion coefficient Ds of the solute in the film. With thick films and smaller diffusion coefficient, analyte molecule travel slower. As a result, slower rate of mass-transfer and an increase in plate height.

Application of Chromatography Qualitative analysis Quantitative analysis

Qualitative Analysis Based on retention time Provided the sample produce the peak at the same retention time as a standard under identical conditions

Quantitative analysis Analysis based on Peak Height The height of chromatographic peak is obtained by connecting the base lines on either side of the peak by a straight line and measuring the perpendicular distance from this line to the peak. Analysis based on Peak Area Peak areas are usually the preferred method of quantitation since peak areas are independent of broadening effects. Most modern chromatographic instruments are equipped with computer or digital electronic integrator that permit precise estimation of peak areas.

Calibration Method (also known as external method) - Involve preparation of series of standard solutions that approximate the composition of the unknown. - The peak heights or areas are plotted as a function of concentration. - The concentration of the component(s) to be analysed is determined by comparing the response(s) peak(s) obtained with the standard solutions.

Internal Standard Method - Equal amounts of an internal standard substance is introduced into each standard and sample. - The internal standard should not react with the substance to be examined; it must be stable and must not contain impurities with a retention time similar to that of the substance to be examined. - The concentration of the substance to be examined is determined by comparing the ratio of the peak areas (or heights) due to the substance to be examined and the internal standard in the test solution with the ratio of the peak areas (or heights) due to the substance to be examined and the internal standard in the standard solution.

Area Normalization Method In the normalization method, the areas of all eluted peaks The percentage content of one or more components of the substance to be examined is calculated by determining the area of the peak(s) as a percentage of the total area of all the peaks, excluding those due to solvents or any added reagents and those below the disregard limit.

TAILING AND FRONTING OF CHROMATOGRAPHIC PEAKS A common cause of tailing and fronting is a distribution constant that varies with concentration. Fronting also arises when the amount of sample introduced onto a column is too large.

TYPES OF COLUMN There are two types of column: Packed Column Capillary Column

PACKED COLUMN Packed column Modern packed columns are fabricated from glass or metal tubing. These tubes are densely packed with uniform, finely divided packing material or solid support, coated with a thin layer (0.05 to 1 µm) of the stationary liquid phase. The tubes are usually formed as coils.

CAPILLARY COLUMN Capillary column Also known as open tubular column

Early wall –coated open tubular (WCOT) column were constructed of stainless steel, aluminium, copper or plastic.later, they are made from glass. The most widely used capillary columns are fused-silica wall-coated (FSWC) open tubular columns. Column constructed of fused silica tubing. Polyimide coating gives it strength (outer layer). This resulting columns are quite flexible and can be bent into coils. These capillaries have much thinner walls compared to glass columns. Liquid stationary phases coated or chemically bonded. Most applications utilize FSWC open tubular column (replacing WCOT glass column).

Recently, 530-µm capillaries, sometimes called megabore column have appeared on the market. These columns maintain the features of capillary column. These columns tolerate sample sizes that are similar to those for packed column. However, the resolution with these columns is lower compared to capillary column (resolution is higher with column of smaller inner diameter). Advantage of megabore column over packed column is their lack of bleeding (loss of stationary phase with time).