1. Instrumental Analysis (I)  HPLC Tutorial #7 PHCMt561 – 5 th Sem. Pharm.

2. Objectives 1. Define theoretical plate number and determine the influence of flow rate and particle size on it (van Deemter equation). 2. Describe the elementary approach to develop HPLC methods. 3. Choose a suitable column size in order to obtain a given efficiency in separation 4. Choose a suitable stationary phase for certain chromatographic separation using a flow-chart 5. Predict changes in elution time or elution order of analytes for a given chromatographic system.

3. 4. 1. Why do bands appear in chromatograms? http://www.studyhplc.com/hplcinstrument.php Ideally, an infinitely narrow band applied at the inlet of a column emerges with a “ Gaussian shape” at the outlet. Main cause for this is diffusion. Each individual mechanism contributing to broadening produces a standard deviation  I, the observed variance (  obs 2 ) of a band is the sum of the variances from all contributing mechanisms. © Dr. Rasha Hanafi, GUC3 Lecture 9 – Chromatography, 28-11- 2010

4. 4.2. Theoretical Plates N 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 HETP (the smaller the better). If the length of the column is L, HETP = L / N Can you think how to decrease HETP of a column? © Dr. Rasha Hanafi, GUC4 Lecture 9 – Chromatography, 28-11- 2010

5. 4.3. Influence of the Flow Rate on HETP © Dr. Rasha Hanafi, GUC5 Lecture 9 – Chromatography, 28-11- 2010 Optimum flow rate

6. 4.3. van Deemter Parameters: A and B  A: Multiple Paths: Some flow paths are longer than others, molecules entering the column at the same time on the left are eluted at different times on the right. Another expression: Eddy diffusion. The smaller the stationary phase particles, the less serious this problem is - it does not occur in open-tubular columns.  B/u: Longitudinal Diffusion: Solutes continuously diffuse away from the concentrated center of their zone. The greater the flow rate, the less time is spent on the column and the less longitudinal diffusion occurs. © Dr. Rasha Hanafi, GUC6 Lecture 9 – Chromatography, 28-11- 2010

7. 4.3. van Deemter Parameters: C  C*u: Equilibration Time ("mass transfer"): The finite time required for solute to equilibrate between the mobile and the stationary phases gives rise to the term C*u in the van Deemter equation. The slower the linear flow, the more complete equilibration is, and the less zone broadening occurs. There may also be zone broadening originating from factors outside the column: 1. When the solute is applied in large volume, i.e., the zone at the starting point is too broad. 2. When the detector flow cell is too large. 3. When the tubing connections are too wide before the column or the detector. © Dr. Rasha Hanafi, GUC7 Lecture 9 – Chromatography, 28-11- 2010

8. 4.4. Influence of Particle Size  Efficiency of a packed column increases as the size of the stationary phase particles decreases. © Dr. Rasha Hanafi, GUC8 Lecture 9 – Chromatography, 28-11- 2010 Under optimum conditions: t R : retention time. w: width of the peak at the base

9. 4.5. High Performance = High Pressure? The smaller the particles of a packed column, the more pressure has to be applied to obtain a certain flow rate: Pressures of approx. 7 – 40 MPa (70 – 400 bar) are required for flow rates of 0.5 to 10 mL/min. 1.More uniform flow when particles are smaller: multiple path A term is reduced 2.Diffusion distance of solute in mobile phase is in the order of particle size: the smaller the particles, the smaller the diffusion distance (C term is reduced) © Dr. Rasha Hanafi, GUC9 Lecture 9 – Chromatography, 28-11- 2010

10. Choose a Suitable Column # 14 1.Estimate the length of a column required to achieve 1.0 x 10 4 theoretical plates if the stationary phase particle size is 10.0 m, 5.0 m, or 3.0 m. 2.Why do smaller particles give better resolution? N  3500 x L [cm] / d p [m]  L 1 [cm]  N x d p [m] / 3500 = 100'000 / 3500 = 28.6 cm.  L 2 [cm]  50'000 / 3500 = 14.3 cm, L 3 = 8.6 cm. more uniform flow through the column (van Deemter A term is reduced) more surface area => better (faster) equilibration (C term reduced)

11. Flow Chart I: Small Molecules 1. m. wt. 2. solubility

12. 1. m. wt. 2. solubility Flow Chart II: Large Molecules

13. Choose the Suitable Method # 13 Suggest the type of liquid chromatography you could use to separate compounds in each of the following categories: 1.FM = 164.5, soluble in octane 2.FM = 482.9, soluble in chloroform 3.FM = 2540, soluble in water, ionic 4.FM = 4205, soluble in THF bonded RP chromatography NP chromatography (adsorption) ion-exchange chromatography depending on molecular size: bonded RP ( 30 nm) chromatography

14. RP Chromatography # 12 Nonpolar aromatic compounds were separated by HPLC on a octadecyl bonded phase. The eluent was 65% vol. MeOH in water. 1.How would the retention times be affected, if 90% MeOH were used instead? 2.Octanoic acid and 1-aminooctane were passed through the same column described above, using an eluent of 20% MeOH / 80% buffer (pH 3.0). State which compound is expected to be eluted first and why. Shorter - on RP stationary phases eluent strength of mobile phase increases as polarity decreases. 90% MeOH/H 2 O is less polar than 65% MeOH/H 2 O, so elutent strength is stronger, so elution is faster. The amine is eluted first. At pH 3 (acid) it is protonated = ionic = polar while the carboxylic acid is generally associated (not deprotonated) = not ionic = less polar. Generally in RP chromatography: the less polar the analytes, the higher their affinity to the stationary phase, the more they are retained.

15. SUMMARY Initially, pressure was selected as the principal criterion of modern liquid chromatography and thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an unfortunate term because it seems to indicate that the improved performance is primarily due to the high pressure. This is, however, not true. In fact, high performance is the result of many factors: very small particles of narrow distribution range and uniform pore size and distribution, high pressure column slurry packing techniques, accurate low volume sample injectors, sensitive low volume detectors and, of course, good pumping systems.