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Chapter 4


Lesson Outcomes:
 Identify and explain the different types of chromatographic

 Explain the principles of gas chromatography, high
performance liquid chromatography, ion exchange and size
exclusion and etc.
 Interpret analytical information from chromatographic data
 Recognize problems arising from resultant spectrum and
chromatograms and suggest probable solutions

4.1 A General Description of


 Is a technique used to separate and identify the components of a mixture  Works by allowing the molecules present in the mixture to distribute themselves between a mobile and a stationary phase mobile phase = solvent or gas stationary phase = column packing material 4 .

components that are weakly held by the stationary phase travel rapidly (fast) As a consequence of these differences in mobility. sample components separate into discrete bands that can be analyzed qualitatively and/or quantitatively .How separation occur?  Chromatography used to separate and identify the     5 components of complex mixtures Works by allowing the molecules in the mixture to distribute themselves between a stationary and a mobile phase Components that are strongly retained by the stationary phase move slowly with the flow of mobile phase In contrast.

Either gas.Classification of chromatographic methods 6 1. .The way stationary and mobile phases are brought into contact. Based on the kinds of equilibria involved in the in solute transfer between the phases -Interaction of analyte between stationary and mobile phases. liquid or supercritical fluid. 2. 3. Based on the types of mobile phase . Based on physical means .

Supercritical-fluid chromatography (SFC) 7 Planar Chromatography  stationary phase is supported on a flat plate or in the interstices of a paper.Chromatographic Methods based on physical means Column Chromatography  stationary phase is held in narrow tube. mobile phase moves through capillary action or gravity  Example: Thin-layer chromatography (TLC). Paper chromatography (PC) . mobile phase moves by pressure or gravity  Example: Gas chromatography (GC).

Column Chromatography 8 Planar Chromatography .

Chromatography based on types of mobile phase Column chromatography can further be differentiated based on the types of mobile phase. Gas  eg: gas Chromatography Liquid  eg: liquid chromatography 9 Supercritical fluid  eg: supercritical fluid chromatography .

Gas Chromatography 10 Liquid Chromatography Supercritical Fluid Chromatography .

Classification of Chromatographic Methods Chromatography Partition Liquidliquid 11 Gasliquid Adsorption Liquid-solid Gas-solid Ion-exchange Liquid-solid Size-exclusion Liquid-solid .

Cation – analyte is cation.Anion – analyte is anion. the mobile phase polar 12 .Normal – stationary phase polar. bonded phase has negative charge  Partition – based on the relative solubility of analyte in mobile and stationary phases .Reverse – stationary phase nonpolar. the mobile phase nonpolar . bonded phase has positive charge .Types of chromatography on the basis of interaction of the analyte with stationary phase  Adsorption – for polar non-ionic compounds  Size Exclusion – stationary phase is a porous matrix sieving  Ion Exchange – for ionic compounds .

13 Size Exclusion – separate molecules by size.stationary phase is a porous matrix .Chromatography based on interaction of the analyte with stationary phase 4. sieving.

Stationary phase: solid Mobile phase: gas/liquid .of solute on surface of stationary phase.Adsorption Chromatography     14 Adsorption . the components of the mixture stick to its surface with varying degrees of strength and thus separate. for polar non-ionic compounds 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.

TLC Plates 15 .

Separations of the components will be based on relative solubility of the components in the mobile and stationary phase  Partition . Normal phase – stationary phase polar. the mobile phase polar 16 .Partition Chromatography  Partitioning is a distribution (by dissolving) of the components between 2 immiscible phases.based on the relative solubility of analyte in mobile and stationary phases a. Reverse phase– stationary phase nonpolar. the mobile phase nonpolar b.

Polar components will retain longer than the non- polar components ii. Non-polar components will move quickly through stationary phase and will elute first before the polar components and vice-versa  The stationary phase actually consists of a thin film adsorbed (stuck) on or chemically bonded to the surface of a finely divided solid particles  If the mobile phase is gas. the volatility (vapor pressure) and solubility in stationary phase plays an important role 17 . i. Example of partitioning using polar stationary phase.

18 .

Cation – analyte is cation.attraction of ions of opposite charges. i.Ion Exchange Chromatography  Method for separating mixture of ions  Ion Exchange . bonded phase has negative charge  Sample is aqueous solution of inorganic ions or organic ions  Stationary phase are 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 19 through it. Anion . bonded phase has positive charge ii.analyte is anion. for ionic compounds i.

The mobile phase is then change for several times in a stepwise fashion so that one kind of ion at a time is removed. Ions that bond to the charged site on the resin bead     20 are separated from organic or inorganic ions aqueous solution. The process is repeated until complete separation achieved. The process is repeated several times by changing of the mobile phase composition The process begin with initially running the analysis using a mobile phase with all the ions in the mixture. .

21 .


Size Exclusion Chromatography
 Size Exclusion – separate molecules by size;

sieving- stationary phase is a porous matrix
 Also called gel permeation chromatography.
 Separation technique of dissolved species is based
on the size of the components.
 Stationary phase: porous polymer resin particles
(molecular sieves).
 The components to be separated enter the pores
of these particles and are slowed down from
progressing through this stationary phase.

 Separation depends on the sizes of the pores

relative to the sizes of the molecules to be
 Small particles are retarded to a greater extent
than large particles (some of which may not
enter the pores at all) and separation occurs.
 Particles with size bigger than the pore size will
be eluted first from the column


25 .

A mobile phase then passes over or through the stationary phase  Retention time: Time required for the sample to travel from the injection port through the column to the 26 detector .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.

2 Migration Rates of Solutes 27 .4.

K (partition constant) is the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase [ A]stationary K [ A]mobile 4.1 Distribution Constant  An analyte is in equilibrium between 2 phases.2. Amobile ↔ Astationary  The equilibrium constant. k’ (capacity factor) is used to describe the migration rate of an analyte on a tR  tM column 28 k' A  tM .2 Retention Factor  Retention factor.4.2.

2.4.3 Retention Time  Retention time (tR) is the time for the analyte to pass through the column of the time between the sample injection and an anlyte peak reaching a detector at the end of the column 29 .

2.5 Adjusted Retention Time  The adjusted retention time (tR’) is the time a compound spends in the stationary phase  tR’ is the difference between the retention time (tR) and the dead time (tM) for a compound 30 tR '  tR  tM .4.2.  tM can be expressed as the time required for an average molecule of the mobile phase to pass through the column 4.4 Dead Time  Dead time (tM)is the time a non-retained compound spends in the mobile phase (the amount of time the non-retained compound spend in the column.

the more separation between two compound or peaks k' B  k' A *Note that species A elutes faster than species B 31 .4. the compounds cannot be seperated  The higher the selectivity.6 Selectivity Factor  Selectivity factor α) is the ratio of the capacity factors of two peaks which describes the separation of two species (A and B on the column)  The selectivity is always greater than one (α>1)  If the selectivity equals to one (α=1).2.

3 The Efficiency of Chromatographic Column 32 .4.

thus more compounds can be separated by the column and vice versa 33 . milimeters)  The efficiency increases as the height of theoretical plate decreases.Description of Column Efficiency  The efficiency is related to the number of compounds that can be separated by the column  The efficiency is expressed as the number of theoretical plates (N. unitless) or as the height equivalent to a theoretical plate (HETP.

The Theoretical Plate Model of Chromatography  The plate model proposes that the chromatographic column contains a large number of separate layer (theoretical plates)  The analyte moves down the column by transfer of equlibrated mobile phase from one plate to the next The column H Theoretical plates 34 *Note that the plates do not really exist. they’re just figment of imagination in order to understand the separation process in the column .

5tR N 2 w1 2 2 Where.Where. w = w1/2 is the peak width at half-height . H = height of the plate L= length of the column N= number of theoretical plate L H N  The number of theoretical plates that a real column can be found by examining a chromatographic peak after elution 2  tR  N  16  W  or 35 5. W = width of the peak Where.

4.4 Column Resolution 36 .

1 The Effect of Retention and Selectivity Factors on Resolution  Resolution is the measure of how well species have been separated 2[(tR )B  ( tR )A] WA  WB Where. ΔZ = separation btwn peak A and B WA and WB = widths at the base of peaks A and B 37 .4.4.

0 and the best resolution between two peaks requires Rs>1.5  To relate with the number of plates (N). the selectivity factor (σ) and the retention factors (k’) of the two solutes. Acceptable resolution is Rs=1. N R 4    1  k ' B        1  k ' B  Simplified: Rs = √ N 38 .

Rs values of less than 1.0 are considered unresolved peaks 39 .

4.2The Effect of Resolution on Retention Time  Relationship btw the resolution of a column and retention time 16 R H    (1  k ' B )3 (tR ) B        1  (k ' B ) 2 2 S 2 Where.4. Ʋ = velocity of the mobile phase Simplified: tR = Rs2 40 .

H iV) length of column to achieve Rs 1. Rs ii) the average number of plates. N iii) the average plate height.30 min Length of column: 30 cm Peak widths: A = 1.Example: 17.5 41 .40 min 1.21 min Calculate: i) column resolution.11 min B = 1.63 min 16.

7 x 10-3 cm = 60 cm .44 x 103 = 8.06 = 3.Answer: i) 2[(tR )B  (tR )A] Rs  WA  WB ii)  tR  N  16  W  iii) L H N 2 iv) (Rs)1 = √N1 (Rs)2 √N2 42 = 1.

3.Variables Affecting Column Efficiency 1. 4. 2. 43 Mobile phase flow rate Particle size Diameter of column Film thickness .

Effect of Mobile Phase Flow  From both the plots for LC and GC. we can see that both show a minimum in H at low linear flow rates  H increases as the mobile phase flow rate increases 44 .

Effect of Particle Size Effect of particle size on plate height for a packed GC column  The numbers to the right is the packing particle 45 diameters  The smaller the particle size. the more pressure is needed to push the mobile phase through the column  H increases as the particle size increases .

Effect of Diameter of The Column  For packed column. the most important variable that affect column efficiency is the diameter of the particles that making up the packing  The increase in column diameter will increase the plate height 46 .

Effect of Film Thickness  With thick films makes the analyte molecule travel slower. thick films will increase in plate height 47 . As a result.

5 Applications of Chromatography 48 .4.

Qualitative and Quantitative Analysis 25.6 min  Retention time tell as about compound identity = qualitative  Peak Area or height tell us how much of compound is there = quantitative 49 .

Provided the sample produce the peak at the same retention time as a standard under identical conditions 50 .Qualitative Analysis  Based on retention time .

Same compound due to the same tR 51 .

. Suitable if the peak is very narrow and have uniform shapes or when peak area values are not available. Analysis based on Peak Height   52 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.Quantitative Analysis 1.

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.2. A = peak height (h) x w1/2 w1/2 = width at ½ height 53 .

3. The peak heights or areas are plotted as a function of concentration. . Calibration method (also known as external method)    54 Involve preparation of series of standard solutions that approximate the composition of the unknown. The concentration of the component(s) to be analysed is determined by comparing the response(s) (peak(s)) obtained with the standard solutions.

the concentration of analyte can be determined.4. Internal Standard Method  Internal standards are widely used in chromatography because the small quantity of sample solution injected into the chromatograph is not very reproducible in some experiments.  Internal standards are also desirable when sample loss can occur during sample preparation steps prior to analysis. 55 .  By knowing the concentration of the standard compound and its relative response.

  Calibration involves plotting the ratio of the analyte signal to the internal-standard signal (Area ratio or height ratio) as a function of the analyte concentration of the standards Signal from analyte is compared with signal from the internal standard to find out how much analyte is present Area Ratio = Peak Area/Height Analyte Peak Area/Height internal standard Area or height ratio Concentration of analyte in sample 56 Concentration of analyte in standard solution .

5. Analysis based on Peak Area using Response Factor Method  A measure of relative mass spectral respond of an analyte compare to each internal standard  The response factor for compound X can be calculated using the following equation: Response factor = 57 Peak Area of Compound X Concentration of Compound X .

Answer: Step 1: Calculate the Response Factor Response Factor for benzene = 100000 = 50 2000 Step 2: Concentration of benzene in sample = 57000 = 1140 µg/mL 50 .000. An injection of the sample with the unknown concentration of benzene has a peak area of 57. Calculate the concentration of benzene present in the sample.000. Example: 58 An injection containing benzene at a concentration of 2000 µg/mL is made and results in a peak area of 100.

Area Normalization Method  In the normalization method. C: Ci = Ai x 100 AT Ai = Area of component peak in the chromatogram AT = Total area of the peaks in the chromatogram 59 . excluding those due to solvents or any added reagents and those below the disregard limit  Content of a component in the sample.6. the areas of all eluted peaks is normalized  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.

Y and Z.43 1866 Y 650 34. X. Answer : By assuming the response factor of the three compounds is identical. Example: A mixture contains only three compounds. the total peak area and the percentage of each compound can be calculated.43% X 232 12. 650 and 984 arbitrary units.73 TOTAL 1866 100 . Calculate the percentage of each compound. Sample calculation: Compound Peak Composition Area (%) 232 x 100 = 12.83 60 Z 984 52. An injection of 1 µL of the mixture resulted a chromatogram with peak areas of 232.

 Fronting also arises when the amount of sample introduced onto a column is too large. 61 .Tailing and Fronting  A common cause of tailing and fronting is a distribution constant that varies with concentration.

Thinner liquid stationary phase 62 . Narrower columns 3. Lower temperature (GC) 4. Smaller particle 2. Band broadening can be reduced by using: 1.