Chromatography Lecture 3 PDF

Chromatography Lecture 3 Last lecture I promised to recap on how pH impacted elution times  Under reverse phase chromatography, which compound would elute later if the pH was reduced from 7.0 to 5.5 NH 2...

Chromatography Lecture 3 Last lecture I promised to recap on how pH impacted elution times  Under reverse phase chromatography, which compound would elute later if the pH was reduced from 7.0 to 5.5 NH 2 O OH O A.A B.B C.C A B C D E D.D E.E Chromatograms If a detector that responds to solute concentration is placed at the end of a column, and its signal plotted as a function of time you will obtain a chromatogram. Each peak represents a separated component of the mixture. The chromatograph gives the analysts several important details regarding the separation and identification of the components of the mixture. Retention time: tR Retention time: The time taken for a solute to reach the detector after injection (to). Determining how good the chromatographic separation is. Chromatograms can be used for qualitative and quantitative analysis – but before this can be done you need to know “how good” the chromatogram is. Often the following terms are used: Resolution Efficiency 1. Resolution This is a measure of how well the peaks have separated. Resolution is needed to be able to use peak area quantitatively. Resolution (Rs) = twice the distances between the two peak maxima divided by the sum of base width of the two peaks. ~1.5 = good base line separation ~1.0 = resolution not fully complete (only about 90%) 30,000 theoretical plates/m. Which of these 15 cm columns meets the specifications? a)Retention time of analyte 6.4 min, Wh 0.2 min. 2 5.54 x (t R ) Efficiency (n) = 37819.7 plates/m n (b) Retention time of analyte 5.6 min, Wh 0.2 min L x (Wh ) 2 Efficiency (n) = 28955.7 plates/m (c) Retention time of analyte 10.6 min, Wh 0.6 min. Efficiency (n) = 11527.3 plates/m Therefore, only column 1 meets the specifications. A standard operating system states that a chromatography column must have an efficiency of >20,000 theoretical plates. Which of the following 20 cm columns meets this criteria? A. Retention time of analyte 10.6 mins, Wh 0.5 mins B. Retention time of analyte 8.5 mins, Wh 0.5 mins C. Retention time of analyte 15.6 mins, Wh 0.5 mins D. Retention time of analyte 9.8 mins, Wh 0.5 mins E. None of the above 5.54 x (t R ) 2 n 2 L x (Wh )  Factors effecting efficiency, causing band broadening in HPLC. Chromatographic peaks have widths, so identical molecules are taking different lengths of time to travel through a column. This is undesirable, and we aim to have peaks as sharp as possible. The more rapidly a peak broadens the less efficient the column is. So why does this occur and what factors effect it? 1. Multiple path effect Packaging of a column is not perfectly uniform; therefore. a solute molecule could take a different path through a column and travel through at different speeds. This is known as the multiple path effect, and lead to band broadening.  2. Non-equilibrium mass transfer Normally when a solute is partitioned between a mobile and stationary phase it is near the surface of both, but not always. By random chance, some molecules may travel further into the stationary phase, or be too far away from the surface of the stationary phase to partition at any one moment in time. This leads to variable times for partitioning in and out of the stationary phase.  3. Sample size Ideally, the sample size should have no effect on the ratio of solute in the stationary phase compared to the mobile phase and as you increase solute concentration, CS and CM should increase proportionally. This will lead to a perfect Gausian peak. However, if you increase the solute concentration too much, the stationary phase may become saturated. This leads to a tailing peak. 4. Diffusion Diffusion is the movement of solute molecules from high concentration to low concentration. Therefore, it works to broaden peaks even when the mobile phase is flowing. Diffusion occurs mostly in the mobile phase and is grater in gas chromatography, compared to liquid chromatography.  5. Time on column The longer a compound is on the column the more opportunity the molecules have had to diffuse. The random effects of non-equilibrium mass transfer are also more apparent over time since there have been many more partitioning events occurring. Hence you will tend to see peaks broaden over time. Self-test: Indicate which of the following parameters can decrease or increase column efficiency in liquid chromatography: Very low flow rate D Low diffusion coefficient in the mobile Large particle size of stationary phase D phase D Small particle size of stationary phase High diffusion coefficient in the mobile I phase Thick stationary phase coating D I Low diffusion coefficient in the stationary Thin stationary phase coating I phase D Regularly shaped particles of stationary phase I High diffusion coefficient in the Irregularly shaped particles of stationary phase stationary phase D I High temperature I Low Temperature D Uneven Stationary phase coating D Even stationary phase coating I Overall factors required for high efficiency (narrow bands): Small sample size – reduces tailing and fronting. Small particle size – Minimises contribution to band broadening due to the multiple path effect, and reduces the effect of the non-equilibrium mass transfer. Thin layer of stationary phase – Minimises the non-equilibrium mass transfer. Optimum mobile phase velocity – To fast and there will not be time to separate, too slow and peaks will vriadern over time. Low dead volume – This is areas where partition cannot happen e.g. the space between injection and the start of the column, or end of the column and the detector. This is undesirable because it allows for more diffusion. Which of the following is most likely to improve the efficiency of chromatographic peaks? A. Minimising non equilibrium mass transfer B. Increase the sample concentration C. Increase the variability in the size and shape of the particles making up the column. D. Encourage the multiple path effect E. Slow down the flow rate of the column  End Move to Lecture 4 - Chromatography