In this slide contains Factors Affecting Resolution In HPLC and its criteria's. Presented by: M.Sudheeshna. (Department of pharmaceutical analysis). RIPER,anantpur.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M.Pharm I Semester
Presented by
M.SUDHEESHNA (Reg.no:20L81S0713)
Pharmaceutical analysis
Under the guidance of
Dr. P.Ramalingam M.Pharm, Ph.D.
Director – R&D Division,
Professor of pharmaceutical analysis and medical chemisry
President – IPA local branch - anantapuramu
FACTORS AFFECTING RESOLUTION IN
HPLC

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction
• Factors affecting resolution in HPLC
 Capacity factor , K’
 Selectivity factor , α
 Column efficiency , N
 Tailing factor ,T
• Acceptance criteria
• How to improve resolution
• References
Contents:

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• HPLC is characterized by the use of high pressure to push mobile phase solution
through a column of stationary phase allowing a separation of complex mixtures
with high resolution.
• They travel according to their relative affinities towards stationary phase.
• More affinity – travels slower
Less affinity - travels faster
• This technique is used for separation , purification, identification, extraction of
compounds.
Introduction:

4. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Resolution is a function of difference in retention times of the two
peaks (t RA and t RB) and widths of the peaks at the base (W A and
W B).
• Baseline resolution is achieved when R = 1.5.
• There is a quantitative measure of resolution as shown in the
equation below:
R = t RA – t RB / 0.5( WA + W B)
Resolution:

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Resolution can also be expressed in the Resolution Equation as a
combination of the factors (separation, efficiency, and retention) that
affect this value.

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
• Retention factor / capacity factor is the time the component
interacts with the stationary phase (tR ) relative to the time the
component spends in the mobile phase (t0).
• It is a measure of degree of interaction with stationary phase.
K= (tR–t0 ) / t0
• Where, tR = retention time of the peak
t0 = column dead time
Capacity Factor, K’:

9. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
Influencing the capacity factor:
• Mobile phase strength : Increasing mobile phase strength – decreases k
• Temperature : Increase in temperature - decreases k

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• As k grows longer its effect reaches a limit at a value of about 10.

11. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• The separation factor is defined as the ratio of retention
factors (k).
• In addition to resolution, the separation factor (α) is also used as an
indicator of the separation of two peaks.
α = K 2 /K 1
• A good selectivity for HPLC is 1.1, which allows a resolution of 1.5
to be achieved.
Selectivity Factor, α

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
Influencing the selectivity factor , α :
• Mobile phase type : type of interaction between m.p and analyte is
critical to optimization of selectivity of system.
• Column type : bonded phase - better selectivity

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• As α grows larger, its effect reaches a limit at a value of about 5.

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• The efficiency of a column is reported as the number of theoretical
plates (plate number), N.
• It is the measure of sharpness of the peaks.
• A high value for efficiency indicates that more peaks can be
separated.
• The number of plates will increase with the length of the column.
Column Efficiency, N

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
• The calculation for efficiency is related to the peak width and is as follows:
N = (t/w) 2
• Where, t = retention time of the peak of interest
W = peak width at the base.
• Similar to the measurement for resolution, the measurement for efficiency
may also be performed using the peak width at half height:
N = 5.54 (t / wh/2 ) 2

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• where, W h/2 is the peak width as half height.

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• Resolution will continue to increase as theoretical plates increase.

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19. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• It is defined as the distance between the front edge of the peak to the
back edge of the peak divided by twice of the front edge of the peak
with all measurements made at 5% of maximum peak height.
• Tailing factor is a measure of symmetry of a peak.
Tailing Factor, T:

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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• It is measured by the following equation:
T = W 0.05 / 2f
• where, W 0.05 = peak width at 5% height
f = distance from peak front to apex point at baseline

21. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Acceptance Criteria:

22. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
Increase N (efficiency) by:
• Increasing column length
• Decreasing particle size
• Reducing peak tailing
• Increasing temperature
• Reducing system extra-column volume
How to improve resolution in HPLC

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
Change α (selectivity) by:
• Changing column stationary phase
• Changing mobile phase pH
• Changing mobile phase solvent(s).
Increase k (retention) by:
• Using a weaker solvent (changing polarity)
• Changing the ionization (polarity) of the analyte by changing pH
• Using a stronger stationary phase (changing polarity)

24. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
References:
• K., Robards (1994). Principles and practice of modern chromatographic
methods. Haddad, P. R., Jackson, P. E. Amsterdam: Elsevier/Academic
• Y. V. Kazakevich and R. LoBrutto (ed.), HPLC for Pharmaceutical Scientists,
2007.
• Lindsay, S.; Kealey, D. (1987). High performance liquid chromatography.
• Patel A, Dwivedi N, Kaurav N, Bashani S, Patel S, Sharma HS, et
al. Chemical analysis of pharmaceuticals: a review. J Med PharmInnov
2016;3:4-7.

25. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25