Hplc

Peak shapes, capacity factor, selectivity,plate number,plate height,resolution and band Broadening

1. HPLC (Peak shapes, capacity factor, selectivity,plate number,plate height,resolution and band Braodening ) Presented by_Yunes Alsayadi Presented to - Dr.Rahul Kumar M.Pharm(Analysis) 2nd sem, ISF College of Pharmacy, Moga, Punjab

2. Introduction to Peak Shapes in HPLC One of the aims of chromatographers everywhere is to get nice sharp Gaussian peaks for each of the components in the run The ideal is a Gaussian or symmetrical shaped peak, a narrow peak width at half-height when compared to its height and no peak fronting, tailing or broadening. Poor peak shape can cause integration and resolution errors in your analysis — which ultimately means poor analysis and wasted time and money. Have you ever encountered a situation during HPLC analysis when you thought: "something is wrong with these peak shapes."? Abnormal peak shapes are a common problem when conducting routine analysis work. Peak abnormalities that are clearly noticeable in chromatograms include peak broadening (including extreme tailing or leading edges), shoulder peaks, and split peaks

3. Peak Asymmetry Asymmetrical peaks are more difficult to resolve, therefore, integration of the peak to provide a peak area for quantitation will also be much less reproducible. Tailing describes a peak whose tail portion (distance ‘B’ in the diagram) is wider than the front portion (distance ‘A’ in the diagram). Due to the effects of instrument dead-volume, adsorptive effects of the stationary phase and the quality of the column packing, peaks may often show a tailing behavior . Also, if the sample concentration is too high or if the column is damaged and contains ‘channels’ then a fronting peak shape may occur.

4. What is Good Peak Shape and Why is it Important ? • Good peak shape can be defined as a symmetrical or gaussian peak and poor peak shape can include both peak fronting and tailing. Good peak shape can be defined by…. Tailing factor of 1.0 High efficiency Narrow peak width Good peak shape is important for…. Improved resolution (Rs) More accurate quantitation Longer usable column lifetime (based on system suitability criteria)

5. How is Peak Shape Measured? • Measures • USP Tailing Factor – at 5% of peak height* • Asymmetry – at 10% of peak height • Indicators • Efficiency – plates* • Peak Width – peak width at ½ height*

6. Factors Affecting Peak Shape Column packing factors • Silica type • Bonded phase and endcapping Mobile phase factors Sample factors

7. High Purity, Low Acidity Silica Improves Peak Shape Standard Silica High Purity, Low Acidity ZORBAX Rx-SIL

8. Mobile Phase Factors for Improved Peak Shape pH Inconsistent and tailing peaks may occur when operating close to an analyte pKa and should be avoided. Buffers Buffered mobile phases enhance retention, resolution, and peak shape. Organic modifiers Changing the organic modifier may improve peak shape due to secondary interactions. Additional mobile phase(using modifiers (TEA, TFA)

9. pH

10. buffers

11. Ghost Peaks Ghost peaks are contaminant peaks that appear even when no sample is injected. There are many causes for ghost peaks and this note will describe how to troubleshoot these contaminant peaks, when you see them. The primary cause of a ghost peak is a dirty pre-column or column. Remove the pre-column and run a sample. If the ghost peaks are no longer present, replace the pre-column (or frit).

12. Sample and Additional Considerations for Good Peak Shape Injecting in a solvent stronger than the mobile phase can cause peak shape problems, such as peak splitting or broadening. Sample Overload May Cause Peak Fronting and Tailing Dead Volume in Flow Lines Dead volume is the total volume of the liquid phase in the chromatographic column.

13. Guidelines for Improved Peak Shape Select columns based on high purity fully hydroxylated silica – Zorbax Rx-Sil based columns. Select double endcapped columns for mid pH or difficult basic compounds, such as Eclipse XDB. Select wide-pore columns for high molecular weight analytes Use buffered low pH mobile phases to reduce secondary interactions Use additional additives only when needed Check sample solvents

14. Retention (Capacity) Factor (k) The retention (or capacity) factor (k) is a means of measuring the retention of an analyte on the chromatographic column. • Retention time (RT) is the difference in time between the point of injection and appearance of peak maxima. • It is also defined as time required for 50% of a component to be eluted from a column. • It is measured in minutes and seconds . • The retention time is longer when the solute has higher affinity to the stationary phase due to its chemical nature.

15. A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase. The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound . The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0, which is also known as the ‘hold-up time’ or ‘dead time.

16. How to change Retention (Capacity) Factor (k) The most effective and convenient way to alter the retention factor of a peak is to adjust the ‘solvent strength’ of the mobile phase. Characteristically reversed phase HPLC has a non-polar stationary phase, therefore, increasing the polarity of the mobile phase will increasingly repel the hydrophobic (nonpolar) sections of the analyte molecules into the stationary phase and the analyte will be retained for longer on the column. The largest gain in resolution is achieved when the k value is between 1 and 5. k values less than 1 are unreliable as analytes may be eluting with other sample components or solvent. Too much retention wastes valuable analysis time and the chromatographic peak height will decrease as the bandwidth of the peaks increases.

17. SELECTIVITY(SEPARATION) FACTOR (α) The selectivity (or separation) factor (α) is the ability of the chromatographic system to ‘chemically’ distinguish between sample components. It is usually measured as a ratio of the retention (capacity) factors (k) of the two peaks in question and can be visualized as the distance between the apices of the two peaks. By definition, the selectivity is always greater than one – as when α is equal to one, the two peaks are co-eluting (i.e. their retention factor values are identical).

18. High α values indicate good separating power and a good separation between the APEX of each peak. However, the alpha value is NOT directly indicative of the resolution. Selectivity values Separation ≥ 2 Easy separation 1.5-2 Possible separation 1.2-1.5 Difficult separation ≤ 1.2 Very difficult separation

19. Effects of S electivity on Resolution Changing selectivity can have a dramatic effect on the chromatographic resolution  Selectivity is relatively simple to alter, with mobile phase constituents (solvent type, ion pair reagents etc.) and pH being the most frequently used methods of adjustment. If suitable resolution cannot be achieved by altering the mobile phase constituents, an alternative column chemistry should be investigated as a means of altering the selectivity of the separation.

20. Theoretical Plate Number The plate number (N) is a measure of the peak dispersion on the HPLC column, which reflects the column performance. Efficiency is derived from an analogy of Martyn and Synge who likened column efficiency to fractional distillation, where the column is divided into Theoretical PlatesThe efficiency of a column is reported as the number of theoretical plates (plate number). Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column. where, tr is the retention time measured from the instant of injection w is the peak width

21. Poor chromatograms are those with early peaks (small tr) that are broad (large w), hence giving small N values, while excellent chromatograms are those with late-appearing peaks (large tr) that are still very narrow (small w), thereby producing a large N. The number of theoretical plates is a measure of the “goodness” of the column. Plate numbers may range from 100 to 106. The plate number depends on the length of the column. A typical plate number for a 4.6 × 100 mm column with 5 μm particles is between 5000 and 8000. A more appropriate parameter for measuring efficiency is the height equivalent to a theoretical plate (or plate height)

22. Factors affecting column efficiency (plate number) Column length Particle size Packing quality Linear velocity (flow) Instrument quality (dead volume) Retention factor For a given column length, the plate number (Nth) is inversely related to the particle size of the column packing. The smaller the particles, the higher the plate number and the separation power. The plate number is also dependent on the flow rate (F) of the mobile phase.

23. HEIGHT EQUIVALENT OF A THEORETICAL PLATE (HETP) A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. It can also be called as a functional unit of the column. The height of one theoretical plate is referred to as the ‘Height Equivalent of a Theoretical Plate. A theoretical plate can be of any height, which describes the efficiency of separation. If HETP is less, the column is more efficient. If HETP is more, the column is less efficient. HETP = length of the column/ no. of theoretical plates HETP is given by Van Deemeter equation Best known is the van Deemter equation , which describes the various contributions to plate height (H). In this equation the parameters that influence the overall peak width are expressed in three terms:

24. where, H = HETP (plate height) A = eddy diffusion term B = longitudinal diffusion term u = linear velocity C = Resistance to mass transfer coefficient

25. Peak height and peak broadening are governed by kinetic processes in the column such as molecular dispersion, diffusion and slow mass transfer. A-term: eddy diffusion : The column packing consists of particles with flow channels in between. Due to the difference in packing and particle shape, the speed of the mobile phase in the various flow channels differs and analyte molecules travel along different flow paths through the channnels. B-term: longitudinal diffusion : Molecules traverse the column under influence of the flowing mobile phase. Due to molecular diffusion, slight dispersions of the mean flow rate will be the result. C-term: resistance against mass transfer : A chromatographic system is in dynamic equilibrium. As the mobile phase is moving continuously, the system has to restore this equilibrium continuously. Since it takes some time to restore equilibrium (resistance to mass transfer), the concentration profiles of sample components between mobile and stationary phase are always slightly shifted.

26. Resolution The most important thing in HPLC is to obtain the optimum resolution in the minimum time. The resolution of a elution is a quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation. It is defined as the difference in retention times between the two peaks, divided by the combined widths of the elution peaks. Where B is the species with the longer retention time, and tR and W are the retention time and elution peak width respectively. If the resolution is greater than one, the peaks can usually be differentiated successfully.

27. A resolution value of 1.5 or greater between two peaks will ensure that the sample components are well ('baseline') separated - to a degree at which the area or height of each peak may be accurately measured. • Resolution is calculated using the separation of two peaks in terms of their average peak width at the base (t R2 > t R1).

28. Calculate the resolution, R, between two peaks by R = (RT1 - RT2) / [0.5 * (W1 + W2)], where RT1 and RT2 represent the retention times of peaks 1 and 2, and W1 and W2 represent the widths of the peaks taken at their bases. Example, if one peak exhibits a retention time of 16.8 seconds and a width of 3.4 seconds . If the second peak exhibited a retention time of 21.4 seconds with a width of 3.6 seconds , then the resolution would be R = (21.4 - 16.8) / [0.5 * (3.4 + 3.6)] = 4.6 / 3.5 = 1.3.

29. Band Broadening Band broadening is a phenomenon that reduces the efficiency of the separation being carried out –leading to poor resolution and chromatographic performance The degree of band broadening (loss of efficiency) naturally increases with the age of the chromatographic column being used broadening in chromatographic systems can be divided into two broad areas of concern One is the contribution from what is known as dead volume and The other source of broadening is within the column . Dead volume refers to all the volume in a chromatographic system from the injector to the detector other than the column.

30. Broad peaks: Possible cause: Overloading of column Retention time too long Extra-column volume too large "Dead" volume Detector time too slow Possible solution: Dilute sample 1:10 and rerun Modify gradient so that peaks elute earlier Reduce tubing diameters and lengths where possible, particularly post-column Check all connections for proper fit Increase detector Hz rate

31. Peak Tailing: Peak Fronting: Possible cause: Possible solution: Dead volume in flow path Check all fittings, especially post-column and at the column head Mobile phase too weak Remake mobile phase or increase acid concentration Overloading of column Dilute sample 1:10 and rerun or use a larger capicity column Column is poorly packed Run high organic through column, then equilibrate and retry, or replace column

32. Peak abnormalities

33. References https://www.chromacademy.com/lms/sco3/Theory_Of_HPLC_Band_Broadening.pdf https://www.shimadzu.com/an/hplc/support/lib/lctalk/resol-1.html https://www.slideshare.net/shwetamore5/theory-of-high-performance-liquid-chromatography-ppt https://www.agilent.com/cs/library/eseminars/Public/secrets%20of%20good%20peak%20shape%20in%20hplc.pdf https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Analytical_Sciences_Digital_Library/JASDL/Courseware/Separation_Science/02_Text/03_Broadening_of_Chromatographic_Peaks https://sciex.com/community/support-community/faqs-and-discussions/lc-troubleshooting/peak-shape/what-s-the-reason-for-broad-peaks-peak-tailing-or-peaks-fronting