INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS

1. INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS

2. 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.

3. 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.

4. 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

5. Classification of Chromatographic Methods Column chromatography Planar chromatography • Stationary phases is held in narrow tube; mobile phase moves by pressure or gravity E.g. – gas chromatography (GC) – supercritical-fluid chromatography (SFC) • 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) * Based upon physical means

6. 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

7. 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

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

10. Partition Chromatography

11. 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

12. 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.

13. 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.

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

15. Adsorption Chromatography

16. 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

17. Ion-exchange chromatography

18. 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.

19. 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

20. Size-exclusion chromatography

21. 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.

22. 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.

23. Terminologies in chromatography

24. 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

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

26. 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

27. 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.

28. 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.

29. 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 – tMtM 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

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

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

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

33. Column length L H • 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. Plate height Number of theoretical plates Efficient column has small plate height

34. 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

35. Another method for approximating N is to determine W½, the width of the peak at half its maximum height. N = 5.54 tR2 W½

36. 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

38. 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

39. 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

41. 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

42. 17.63 min Response tR 16.40 min tR 1.30 min tM 1 3 6 Retention time , min

43. Rs = 2[tR(B) – tR(A)] WA + WB 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 2 tR W N = 16 2

44. 2 Therefore, calculate the N average Nave = 3.44 x 103 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 =

45. 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

46. 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

47. VARIABLES AFFECTING COLUMN EFFICIENCY

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

50. 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.