Phyto-Analysis Sheet Number : 16 Prof. Dr. Talal Aburjai Page 1 of 9
How to read the chromatogram? Comes from any automated chromatography. The chromatograms show the 0 t (t m ) which indicates the solvent and the peak that represent the analyte. The retention time R t can be calculated, the width of the peak, AUC for quantitative purposes. Corrected R t (t R)=t R m t t R : is the time between injection of the sample till seeing the peak. {Adjusted retention time: An analyte's retention time (tr) minus the elution time of an unretained peak (tm). tr'= tr-tm Adjusted retention time is also equivalent to the time the analyte spends in the stationary phase.}from the internet! W h W b The tr' (is important in manual process) that any delay in the injection time will shift all the peaks. Suppose the injection time was delayed by 1 min, the peak will be shifted 1 min if its t R is 10 it will become 11 while the standard is 10, so this indicate that they are not the same but actually they are the same! So usually we depend on the corrected t R (adjusted) in order to have true value regardless personal or instrumental errors. To understand what is happening inside the column there are some mathematical equations: 1. Capacity factor K (kappa)the ability of the column to separate any compound. K = tr tm = t R tm tm As K increase the separation of the peak from tm is better. Usually if K <1 means the separation of this compound from the front of the solvent is bad (too early). K >5 it is too late. When k' is less than 1.0, separation is poor When k' is > 30, separation is slow When k' is = 2-10, separation is optimum Page 2 of 9
The best value for K is 1-5.(1-10 is acceptable) K >30 too long (very slow). K has nothing to do with other factors either the column nature (normal or reversed). As the length of the column (L) increases the retention time increases. 2. Partition coefficient: the volume of substance in the stationary to the volume of the substance to the mobile phase. This will affect K. 3. Efficiency of the separation: Efficiency is related theoretically to the various kinetic processes that are involved in solute retention (partition coefficient) and their transport inside the column. In order to understand the efficiency of the column: 1.Theoretical plates were created (N). N the efficiency of the separation and vise versa is true. N = 16 ( tr Wb ) 2 The plates number is related to the t R and the peak width W b, inverse relationship between N and W b. In other words; when the column is new and the number of plates is high the peak will be sharp ( W b ) and as the column is consumed the peak will be wide. {Usually with the column there is a sample provided by the manufacturer to test the column, this sample contains reference compounds and all the conditions are determined} N is not related to the peaks like K or α (selectivity factor); it is related to the column. Example: if the column was tested and it has N=20000, after a period of time column is retested and gives N= 5000 that means that there is something happened to the column and this must be corrected. Page 3 of 9
The larger the value of N is for a column, the better the separation of the column between two compounds. - The better the ability to resolve solutes that have small differences in retention. - N is independent of solute retention. - N is dependent on the length of the column. 2. Plate height or height equivalent of a theoretical plate (H or HETP): H N efficiency of separation. 4. Measures of Solute Separation: H = L N Selectivity (separation) factor (α): parameter used to describe how well (efficiency) two solutes are separated by a chromatographic system: α = K a K b α means that the two compounds are well separated. α means poor separation. Why the band spread? [ N, H ( t R or the W b )] 1 In 1 the t R has decreased significantly, while in 2 band broadening occur ( W b ), why?? There are many factors affecting Bands Spread a. Eddy diffusion b. Mobile phase mass transfer 2 Page 4 of 9
c. Stagnant mobile phase mass transfer d. Stationary phase mass transfer e. Longitudinal diffusion a. Eddy diffusion: a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column. 1, 2, 3 are the same substance A, but the way (distant) 1 crossed is not equal to 2 or 3. This is due to the nature of the stationary phase; here it is not well distributed, not well spherical. The stationary phase is not changed it is only fixed; we know that there is something wrong in the stationary phase from the N value tested. Eddy diffusion usually is related directly to the quality of the stationary phase and the column. b. Mobile phase mass transfer: is a process of peak broadening caused by the presence of different flow profile within channels or between particles of the support in the column. Example air bubble trapped in the column will affect the length of the distant crossed by the substance, results in different lengths in the ways of particles for the same substance. The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: 1) The size of the packing material is different 2) The diffusion rate of the solute is not equal at equilibrium. c.stagnant mobile phase mass transfer: band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase). Page 5 of 9
The stationary phase is not well spherical, this will lead to 2 problems 1.the mobile phase mass transfer will not be easily moved or 2.not all the solvent will move in the same way and direction. The degree of band-broadening due to stagnant mobile phase mass transfer depends on: 1) The size, shape and pore structure of the packing material 2) The diffusion and retention of the solute 3) The flow-rate of the solute through the column: F is determined by a valve. If the mobile phase was very fast ( F) lead to Stagnant mobile phase mass transfer; if very slow it will also lead to Stagnant mobile phase mass transfer! d.stationary phase mass transfer: band-broadening due to the movement of solute between the stagnant phase and the stationary phase. (B/c of the shape of the stationary phase) The degree of band-broadening due to stationary phase mass transfer depends on: 1) the retention and diffusion of the solute 2) the flow-rate of the solute through the column 3) the kinetics of interaction between the solute and the stationary phase e.longitudinal diffusion: band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase. The degree of band-broadening due to longitudinal diffusion depends on: 1) the diffusion of the solute 2) the flow-rate of the solute through the column Van Deemter equation: relates flow-rate or linear velocity to H: μ = linear velocity (flow-rate x V m /L) H = total plate height of the column A = constant representing eddy diffusion &mobile phase mass transfer Page 6 of 9
B = constant representing longitudinal diffusion C = constant representing stagnant mobile phase & stationary phase mass transfer All the factors affecting the H are held constant then the relation between H and the flow rate is graphed: There is an optimal flow rate; if the F is or the H will and the efficiency of the separation will. Usually the starting point for any separation using the HPLC is F=1 ml/min. (0.5-1.5 ml/min) Taking into consideration the optimal flow rate we can minimize the effect of A, B, C (the main 3 three factors affecting the separation) The most imp factor among all these factors is the Resolution Rs: to express the good separation between two components. tr2 tr1 Rs = ( Wb2+Wb1 ) 2 In the first picture the resolution is better than the second one. (T R is the imp factor here.) Here the resolution in 1 is =0.75 which is very bad, it is impossible to calculate the width of the peaks, integration here is very difficult. In 2 the resolution was improved Rs=1.0; the first thing to Rs is to K ( t R ), in 3 the resolution is excellent (the t R and the W b are within the range). In general, when Rs> 1.5 is good resolution; Rs<1.0 is considered bad resolution. 1 2 3 Page 7 of 9
K Α N H Rs The ability of Compares two Column Column It is a second the column to separate the peaks Reflect only efficiency efficiency measure of how well two peaks sample retention are separated. If the retention times are the same, the peak width is smaller (sharp) for the one with the larger theoretical plate number, better separation. If the peak width is the same, the retention time is longer for the one with the larger theoretical plate number. N = 16 ( tr Wb ) 2 If the two situations are together, the sharper and longer tr is the best. N is to imagine what is happening within the column. Problems cause band broadening : 1. Injecting complex sample (highly polar or highly nonpolar compounds); it will stick at the head of the column. Mass transfer resistant, N. 2. Trapped air bubbles within the column; degassing is needed. Mass transfer resistant. 3. Sample is not filtered before injection; samples must be filtered by sterile filter (0.25-0.4 μm) b/c larger particles will block the column. Mass transfer resistant. (it is not imp to be sterile) Page 8 of 9
HPLC grade solvents are usually used in columns while analytical grade for other purposes. (The HPLC grade is very expensive b/c it is ultrapure, and is not filtered) 4. Conditioning the column... (conditioning the column with the mobile phase is very important step before stating the separation ) The optimum separation occurs when the two peaks are fully separated from each other, even if the resolution in two different cases was the same. As the capacity factor increase the resolution increase b/c the tr is increased. As the N increase the efficiency of the separation increase. H is decreased. Hiba Tarek عايز تنجز! ركز!!! Page 9 of 9