Akshay ppt

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

Supervised by:
Dr. J. I. Disouza
(Principal)

Tatyasaheb Kore College of Pharmacy, Warananagar

 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

C0MPONENT OF HPLC
 Mobile Phase
 Injector
 In-Line Filter
 Column
 Detector
 Sample
 Pump
 Guard Column
 Connecting Tubing
and Fittings
 Integrator/Recorder
Software

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.

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.

 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.


• 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).

• 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: Height

• 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
column
 Optimize 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)


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.

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
12-Apr-13 T.K.C.P,WARANAGAR.
17/23

 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.

12-Apr-13 T.K.C.P,WARANAGAR.
21/23

 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
specification
monitor 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 8-13
4. 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. 25

12-Apr-13 T.K.C.P,WARANAGAR. 26

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