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this is somewhat helpful article during HPLC study in analyticals.

this is somewhat helpful article during HPLC study in analyticals.

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  • 1. A Seminar On Resolution factor, Telling factor, Theoretical Plates and Capacity factor in HPLC Presented by: Guided by : Mr. Akshay S. Jirage Mr. S. A. Payghan (M.Pharm SEM-I ) (Assistant Professor)Department of Pharmaceutics Department of Pharmaceutics Supervised by: Dr. J. I. Disouza (Principal) Department of Pharmaceutics Tatyasaheb Kore College of Pharmacy, Warananagar
  • 2.  Introduction: What is HPLC? High Performance Liquid Chromatography. High Pressure Liquid Chromatography. High Price Liquid Chromatography.‘’HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.”12-Apr-13 T.K.C.P,WARANAGAR. 1/23
  • 3. C0MPONENT OF HPLC Mobile Phase Injector In-Line Filter Column Detector Sample Pump Guard Column Connecting Tubing and Fittings Integrator/Recorder Software 12-Apr-13 T.K.C.P,WARANAGAR. 2/23
  • 4. Definition:- “High-performance liquid chromatography (or high- pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds, and is used in and analytical chemistry to identify, quantify and purify the individual components of the mixture.”12-Apr-13 T.K.C.P,WARANAGAR. 3/23
  • 5. Terminologies in HPLC Factor:- Retention Time:- The time elapsed between the injection of sample component into the column and their detection is known as the retention time. It is denoted by (tR)12-Apr-13 T.K.C.P,WARANAGAR. 4/23
  • 6. Selectivity Factor(Separation factor):- Relative migration rate of the solute are expressed in terms of selectivity . It is expressed as follows:Where, ky-is distribution constant for the more strongly retained species y. is the distribution constant for the more strongly retained species x.12-Apr-13 T.K.C.P,WARANAGAR. 5/23
  • 7. Column chromatographic efficiency:- It is Height Equivalent to theoretical plate(HETP) It is calculated by following equation HETP=(L/N) Where, L-column length N-Plate countPeak capacity(Pc):-It is the no of peaks that can be separated within a retention window for a specific predetermined resolution.12-Apr-13 T.K.C.P,WARANAGAR. 6/23
  • 8.  Theoretical Plates The concept of the theoretical plate arose from distillation column theory and was borrowed by A J. P. Martin “Chromatographic column contains large number of separate layer called theoretical plate. N, the number of theoretical plates, is used to determine the performance and effectiveness of columns, and is calculated using equation. ・・・(1)• where tr: retention time, and W: peak width12-Apr-13 T.K.C.P,WARANAGAR. 7/23
  • 9. • The following calculation methods are used in actual practice.• 1. Tangent Line Method:-• Peak width is the distance between points where lines tangent to the peaks left and right inflection points intersect the baseline, and is calculated using equation (1). The USP (United States Pharmacopeia) uses this method.• This also presents a problem if the peak is distorted, so that it has multiple inflection points.12-Apr-13 T.K.C.P,WARANAGAR. 8/23
  • 10. • 2. Half Peak Height Method:-• Width is calculated from the width at half the peak height (W0.5). It is the most widely used method. This is the method used by the, BP (British Pharmacopeia), and EP (European Pharmacopeia). 12-Apr-13 T.K.C.P,WARANAGAR. 9/23
  • 11. • 3. Area Height Method:-• Width is calculated from the peak area and height values. This method provides relatively accurate and reproducible widths, even for distorted peaks, but results in somewhat larger N values when peak overlap is significant. ・・・3)• A: Area, H: Height12-Apr-13 T.K.C.P,WARANAGAR. 10/23
  • 12. • 4. EMG Method (Exponentially Modified Gaussian)This method introduces parametersthat accommodate the asymmetry ofpeaks, and uses the peak width at 10 %of the peak height (W0.1). Since it usesa width near the baseline, it results inN values larger than other methods forbroad peaks.12-Apr-13 T.K.C.P,WARANAGAR. 11/23
  • 13.  Resolution(Rs) : Definition:-Resolution describes the ability of column to separate the peaks of interest. Higher the resolution the easier it is to achieve baseline separation between two peaks. Parameter is to be consider here : Efficiency selectivity Retention12-Apr-13 T.K.C.P,WARANAGAR. 12/23
  • 14. • Resolution Factor:-• The position of elution band on the horizontal axis and their widths will determine the extent of separation. The quantitative measure of ability of column to analytes is expressed in terms of resolution factor(R).12-Apr-13 T.K.C.P,WARANAGAR. 13/23
  • 15.  Relative retention is high satisfactory resolution can be obtained. The retention factor ‘k’ is depend only on the ‘strength of the eluent.Resolution Improvement :- ‘K’ value in between (9 and 10) Increase the no of theoretical plate by using longer column Optimize the mobile phase flow rate 12-Apr-13 T.K.C.P,WARANAGAR. 14/23
  • 16. Capacity factor (Retention factor):- capacity factor measures the period of time that the sample component resides in stationary phase relative to time resides in in the mobile phase It is calculated from Retention Time divided by the time for an unretained peak(t0)12-Apr-13 T.K.C.P,WARANAGAR. 15/23
  • 17. Problem arise in analytical work:- Retention factor is much less than unity, elution occur so rapidly that the accurate determination of retention factor is difficult. Retention factor is larger ,somewhere 20-30,the elution times are unusually long. Ideally separation of solute carried out for which the retention factor are between 2 and 10.12-Apr-13 T.K.C.P,WARANAGAR. 16/23
  • 18. Capacity factor (mass distribution ratio,( Dm )• The capacity factor or mass distribution ratio is defined as• This factor determines the retention of a solute and can be calculated from the chromatogram using the following formula:• where• t R = retention time of the solute• t M= retention time of an unretained component12-Apr-13 T.K.C.P,WARANAGAR. 17/23
  • 19.  Tailing factor:- “It is defined as the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope, with all measurements made at 5% of the maximum peak height”.12-Apr-13 T.K.C.P,WARANAGAR. 18/23
  • 20.  Asymmetry factor(As):-• It is defined as the distance from the center line of the peak to the back slope divided by the distance from the center line of the peak to the front slope, with all measurements made at 10% of the maximum peak height. As=b/a where, a=width of front half of peak. b=width of back half of the peak.12-Apr-13 T.K.C.P,WARANAGAR. 19/23
  • 21.  Causes of Tailing Factor:- Deteriorated Column. Flow of mobile phase. Column overload. Incompatibility of sample with Stationary and/or Mobile phase.Why Do Peaks Tail.pdf12-Apr-13 T.K.C.P,WARANAGAR. 20/23
  • 22. Practical application of Tailing factor :-it is important to monitor peak tailing so that itdoes not compromise analytical results.To avoid qualitative and quantitative estimationin LC method.To avoid misinterpretation of peak.12-Apr-13 T.K.C.P,WARANAGAR. 21/23
  • 23.  Practical application Theoretical Plates:-N is usually used to measure and verify the performanceof a column.verify the performance of a column.measure that the resolving power of a new column is tospecificationmonitor the resolving power of a column over time.Determine if column replacement is required due todeterioration in performance.Troubleshoot problems.12-Apr-13 T.K.C.P,WARANAGAR. 22/23
  • 24. References:-1. A article on peak tailing and resolution by John W. Dolan, LC Resources Inc., Walnut Creek, California, USA.2. Practical -High performance Liquid Chromatography by Veronika Meyer 4th Edition publish by John Willy and Sons, pg no14-42.3. The LC Handbook Guide to LC Columns and Method Development By Agilent Technologies. pg no 8-134. Instrumental method of chemical analysis By G .R. Chatwal and Sham k. Anand Himalaya publication pg no 2.624- 2.639.12-Apr-13 T.K.C.P,WARANAGAR. 23/23
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