System Suitability :

Numerical Criteria for the HPLC

Analysis Performance

Efficiency- N
Asymmetry – Af; Tailing - T
Selectivity - α
Resolution - Rs
 Development and Validation of Chromatographic Methods

Quality Control
VIAL SAMPLE NAME INJ VOL No of Inj Function Method Run Time Sample Dilution

System Suitability : 1 Blank

2 System Suitability
20.0
20.0
1
Inject Samples
Inject Samples
LC Demo Method Set
SST Method Set
10.00
10.00
Weight
1.00000
1.00000
1.00000
1.00000

Numerical Criteria for the HPLC 1

Clear
Calibration
LC Demo Method Set

3 Std1 20.0 Inject Standards LC Demo Method Set 10.00 1.00000 1.00000
Analysis Performance 4 Std2 20.0
5
2
Inject Standards LC Demo Method Set 10.00 1.00000 1.00000

Report LC Calibration Report

Report Standard Comparison
Clear LC Demo Method Set

Efficiency- N 1 Std1 20.0

1
Calibration
Inject Standards LC Demo Method Set 10.00 1.00000 1.00000
2 Unk.1 20.0 Inject Samples LC Demo Method Set 10.00 1.00000 1.00000
2
Asymmetry – Af; Tailing - T 3 Unk.2
4 Unk.3
20.0
20.0
2
Inject Samples
Inject Samples
LC Demo Method Set
LC Demo Method Set
10.00
10.00
1.00000
1.00000
1.00000
1.00000
2

Selectivity - α 5 Unk.4 20.0 Inject Samples LC Demo Method Set 10.00 1.00000 1.00000
2
6 Unk.5 20.0 Inject Samples LC Demo Method Set 10.00 1.00000 1.00000
2
7 Unk.6 20.0 Inject Samples LC Demo Method Set 10.00 1.00000 1.00000
Resolution - Rs 1 Std1 20.0
2
1
Inject Standards LC Demo Method Set 10.00 1.00000 1.00000

Clear LC Demo Method Set

Calibration
Calibrate LC Demo Method Set

ABSOLUTE RETENTION OF ONE PEAK Relative Retention Time - RRT

RETENTION FACTOR or CAPACITY RATIO tR,i = RRT
tR - t 0 Cs t R,4
k' =
t0
k’ = φ Cm
RRT<1 RRT>1
1 4 8 10
tR 2 3 6 7 11

VOID

A h B
t0
Referenc
w e Peak

Dr. Shulamit Levin, Medtechnica page 1

 Development and Validation of Chromatographic Methods
PEAK BROADENING c α y2
GAUSSIAN PEAK: c0
= exp (- 2∆T
)
Diffusion while migrating in the column
(y = x0 - wt)

1.0
σ
0.8

0.6 2σ

0.4

0.2
4σ
0.0
0.6065 h = 2 σ x0
0.1353 h = 4 σ
0.8825 h = 1 σ AREA 4σ= 95.6%

PERFORMANCE BY ONE PEAK ADVANTAGES OF HIGH EFFICIENCY

* Better Resolution for peak identification and quantitation
NUMBER OF THEORETICAL PLATES
* Higher Peak capacity for analysis of many components at once
* Higher Sensitivity for detection of low concentrations
tR t
R 2

N= 16 ( ) 1 4
w 8 10
2 3 6 11
7

5
A h t 2
B Or: ( R ) VOID
t0 N = 5.54
w1/2
w

Dr. Shulamit Levin, Medtechnica page 2

 Development and Validation of Chromatographic Methods

PERFORMANCE BY ONE PEAK: PERFORMANCE BY ONE PEAK:

Asymmetric factor or Tailing factor Asymmetric factor or Tailing factor
Symmetric Asymmetric
tR ASYMMETRY FACTOR

B (10% h)
Af =
A (10% h)
TAILING FACTOR
A h h h
B A 5% h + B 5% h
T=
2 X A 5% h
t0
a b a b 5 % of Peak Height
w

Integration Error Caused by Tailing of Peaks PERFORMANCE BY TWO PEAKS

SELECTIVITY FACTOR

k'(2)
T = 1.58
alfa =
T = 1.00 k' (1)
Recovered Peak Areas
Recovered Peak Areas
99.9 % 97.8 % tR(2)
99.8 % 95.3 % tR(1)
99.6 % 92.3 %

t0
w1 w2

Dr. Shulamit Levin, Medtechnica page 3

 Development and Validation of Chromatographic Methods

PERFORMANCE BY TWO PEAKS ADVANTAGES OF HIGH RESOLUTION

EXPERIMENTAL RESOLUTION

t R (2) - t R(1 )
Rs =
1/2 (w 1 +w 2)

tR(2)
tR(1)

t0 0.0 20.0
w1 w2

Higher Components ratio - FACTORS CONTROLLING RESOLUTION

Higher Resolution Required
k' Strength of solvent - polarity
1:1 2:1 5:1 10:1 Strength of packing - surface area, carbon load
Temperature

α Chemistry of solvent - functionality

Rs = 1 Chemistry of packing - functionality
Chemistry of sample - hydrogen bonding or derivatization

N Flow rate - linear velocity Column length

Average particle size Particle size distribution
Column bed configuration Volume of injection
Rs = 1.2 Viscosity of solvent Mass of injection
Temperature (viscosity) Column diameter
Particle shape System dead volume
Viscosity of sample Residence on column
Mixed mechanisms Pellicular vs. porous
Rs = 1.5

Dr. Shulamit Levin, Medtechnica page 4

 Development and Validation of Chromatographic Methods
TYPICAL VALUES N α −1 k’2
Rs = ( ) (
alfa k’ N 4 α k’2+1 )
RESOLUTION 1 0 500
1.1 0.5 1000 5 N=5000
1.2 1 1500 alfa = 1.5 N=4000
1.3 1.5 2000
4 N=3000
N α −1
1.4 2 2500
k’2 1.5 2.5 3000
Rs = ( ) ( ) 1.6 3 3500
3
N=2000
4 α k’2 + 1 1.7
1.8
3.5
4
4000
4500
N=1000
1.9 4.5 5000 Rs 2
2 5 5500
2.1 5.5 6000
Resolution between two peaks is affected by 2.2 6 6500 1
2.3 6.5 7000
the efficiency (N), selectivity (α) and the 2.4 7 7500
0
retention parameter (k’) 2.5
2.6
7.5
8
8000
8500 0 2 4 6 8
2.7 8.5 9000
2.8
2.9
9
9.5
9500
10000
k' (Retention Factor)

TYPICAL VALUES TYPICAL VALUES N α −1 k’2

N α −1 k’2 Rs = ( ) (
alfa Rs = ( ) ( alfa 4 α k’2+1 )
k’ N 4 α k’2+1 ) k’ N
1 0 500 1 0 500 THE MOST SENSITIVE:
1.1 0.5 1000 1.1 0.5 1000 N=5000
10 k' = 9
1.2 1 1500 7 k'=8, 9, 10k'=6 1.2 1 1500
1.3 1.5 2000 alfa = 1.5 1.3 1.5 2000 N=4000
1.4 2 2500
6 k'=4 1.4 2 2500 8
1.5 2.5 3000 1.5 2.5 3000 N=3000
1.6 3 3500 k'=2 1.6 3 3500 N=2000
1.7 3.5 4000 5 1.7 3.5 4000 6
1.8 4 4500 Rs 1.8 4 4500 Rs
1.9 4.5 5000 4 1.9 4.5 5000 N=1000
2 5 5500 2 5 5500 4
2.1 5.5 6000 2.1 5.5 6000
2.2 6 6500 3 2.2 6 6500
2.3 6.5 7000 2.3 6.5 7000 2
2.4 7 7500 2 2.4 7 7500
2.5 7.5 8000 2.5 7.5 8000
2.6 8 8500 2.6 8 8500 0
2.7 8.5 9000 2.7 8.5 9000 1.0 1.5 2.0 2.5
2.8 9 9500 2000 4000 6000 8000 10000 2.8 9 9500
2.9 9.5 10000 N (No of Theoretical Plates) 2.9 9.5 10000 alfa (Selectivity)

Dr. Shulamit Levin, Medtechnica page 5

 Development and Validation of Chromatographic Methods
SELECTIVITY vs EFFICIENCY Separation Development Sequence

tR (2) - t R(1)
Rs = FIRST INJECTION
Same in both cases
1/2 (w 1 + w 2)

ADJUST k' (Retention)

ADJUST N (Plates)

LOW SELECTIVITY (α) HIGH SELECTIVITY (α) ADJUST alpha (Separation)

HIGH EFFICIENCY (N) LOW EFFICIENCY (N)

Methods Development Strategy

Gather Information

Make a Plan

Optimize K’
Incomplete resolution Complete Resolution

Change Alpha

Incomplete resolution Complete Resolution

Optimize N
Incomplete resolution Complete Resolution
Validate Qualitation
FAIL PASS
FAIL

Validate Qualitation
FAIL
FAIL PASS

Evaluate and Optimize method for routine use

- Step by step method development strategy -

Dr. Shulamit Levin, Medtechnica page 6