akshay ppt

Presented by: Mr. Akshay S. Jirage (M.Pharm SEM-I )

Department of Pharmaceutics

Supervised by: Dr. J. I. Disouza

(Principal)Department of Pharmaceutics

Guided by :Mr. S. A. Payghan

(Assistant Professor)Department of Pharmaceutics

Tatyasaheb Kore College of Pharmacy, Warananagar

ASeminar On

Resolution factor, Telling factor, Theoretical Plates and Capacity factor in HPLC

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.”

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Mobile Phase Injector In-Line Filter Column Detector Sample Pump Guard Column Connecting Tubing and Fittings Integrator/Recorder

Software

C0MPONENT OF HPLC


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.”


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)


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. Apr 10, 2023 5/23T.K.C.P,WARANAGAR.


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 countPeak capacity(Pc):-It is the no of peaks that can be

separated within a retention window for a specific predetermined resolution.

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 width


• 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

peak's 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.Apr 10, 2023 8/23T.K.C.P,WARANAGAR.

• 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).Apr 10, 2023 9/23T.K.C.P,WARANAGAR.

• 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: HeightApr 10, 2023 10/23T.K.C.P,WARANAGAR.

• 4. EMG Method (Exponentially Modified Gaussian)This method introduces parameters

that accommodate the asymmetry of

peaks, and uses the peak width at 10 %

of the peak height (W0.1). Since it uses

a width near the baseline, it results in

N values larger than other methods for

broad peaks.


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 Retention


• 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).


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

columnOptimize the mobile phase flow rate


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)

Relative migration rate can be expressed in

selectivity or separation factor.


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. Apr 10, 2023 16/23T.K.C.P,WARANAGAR.

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T.K.C.P,WARANAGAR.

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 component


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”.


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.


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.pdf


Practical application of Tailing factor :-

it is important to monitor peak tailing so that it

does not compromise analytical results.

To avoid qualitative and quantitative estimation

in LC method.

To avoid misinterpretation of peak.


Practical application Theoretical Plates:-N is usually used to measure and verify the performance of a column.verify the performance of a column.measure that the resolving power of a new column is to specificationmonitor the resolving power of a column over time.Determine if column replacement is required due to deterioration in performance.Troubleshoot problems.


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.

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Apr 10, 2023 26T.K.C.P,WARANAGAR.

akshay ppt

Technology

hplc factor

capacity factor

area height method

capacityfactorretention

relative retention

peak area

emg method

peak height w0