HPLC Theory, Method Development

&
Method Validation

Instructors: Dr. Sunil Bhongale & Mr. Dhananjay Sadafule

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 Invention of Chromatography
M. Tswett invented chromatography method in the year of 1906 during his
research on plant pigments. He used liquid-adsorption column
chromatography with calcium carbonate as adsorbent (Stationary phase) and
pet ether as eluent (Mobile phase) to separate chlorophylls/pigments

Ether Chromatography

Chlorophyll
Colors

CaCO3

2
 Classification of Chromatographic techniques depending
on type of Stationary and Mobile phases

Stationary Phase (Solid) Mobile Phase (Liquid or Gas)

Gas Chromatography Liquid Chromatography

Stationary Phase: Solid or Liquid Stationary Phase: Solid

Mobile Phase: Gas Mobile Phase: Liquid

GC: Gas chromatography

LC : Liquid chromatography (or HPLC)

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 HPLC
(High Pressure or High Performance Liquid Chromatography)

Stationary Phase (Solid) Mobile Phase (Liquid)

• The separation depends on the

Mobile affinity/or extent of interaction
phase of analyte towards Stationary &
Strong Weak Mobile phase.

• Thus, the Separation occurs on

Stationary
the basis of varying speed of
phase
solute compounds/analytes that
moves through the column.
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 Chromatographic Elution/Separation
Chromatogram (Detector response, i.e. Absorbance on Y axis and Time on X axis)

(Retention time)

5
Important Parameters for achieving Chromatographic Separation

1. Retention/Capacity factor ( k or k1 )

2. Separation factor (α)

3. Resolution (R)

4. Efficiency of column

(A) No. of Theoretical plates (N)

(B) Height Equivalent To Theoretical Plate (HETP)

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 Retention (or Capacity) factor, k or k1
t R  t0
k
Strength of detector signal

tR t0
tR: Retention time (Peak B)
t0: Non-retention time (Peak A)
t0
(t0 is Retention time of unretained compound
Peak A)

Peak A Peak B
Time

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 Separation Factor (α)
Estimates the extent of separation between adjacent peaks
• Separation factor: Ratio of k’s of two peaks A & B k2

k1 k2 k1
(k 2  k1 )
tR(A)  t0
Where, k 1  For Peak A
t0
tR(B)  t0
Peak A Peak B Where, k 2  For Peak B
t0
tr2-tr1 tr2-tr1

tR(B)  t0

tR(A)  t0
Where, K is Retention or Capacity Factor
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Superior separation Inferior separation
 Resolution, RS
t R 2  t R1
RS 
1
(W1  W2 )
tR1 tR2 2

Wb1
Wb2

Superior separation

W1/2h,1 W1/2h,2 h1/2

Wb1 Wb2
W1 W2
Inferior separation 9
 Efficiency of Column
Theoretical Plate Number (N) & Height Equivalent To Theoretical Plate (HETP)
2
t R 2 .
N  16
tR
5 54
W W1
/2

H Wb1
Wb2
W1/2
H1/2
Superior separation
W

Wb1 Wb2

HETP = L/N
vs Inferior separation 10
 Comparison of Column Efficiency
• Column efficiency is dependent on N Value (the theoretical plate number).
• Columns with high plate numbers (More N Value) are more efficient. A column
with a high N will lead to narrower peak at a given retention time than a
column with a lower N.
• Parameters influencing column efficiency:
– Column length (increasing colum length increases efficiency (increases retention of a peak)
– Particle size (decreasing particle size increases efficiency), reduces peak width of a peak.

N  16( t R
2
tR wb )
tR

wb wb
Larger N Smaller N
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 Method development: To Optimize Parameters for achieving Adequate Separation
K1 = Retention or capacity factor t R  t0 N = 16 (tR/W)2
N = Theoretical pate number k
α = Separation factor t0
k2
 
Case Before k1
adjustment ( k 2  k1 )

k’ increased e.g. Eluent replaced (Mobile phase)

with one of lower elution strength.

e.g. Column replaced with phase having

N increased Particle Size 3 μ instead of 5 μ.

e.g. Mobile phase composition (polarity)

 increased Changed/Column temperature changed.

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Some common Types of HPLC
1. Ion-exchange chromatography : For Ionic samples (Anion & Cation Exchangers phase)

2. Size exclusion chromatography: Used for Larger molecules (St phase with pores)

3. Normal phase chromatography: Polar stationary phase & non-polar mobile phase

4. Reverse Phase chromatography: Non-Polar stationary phase & polar mobile phase

Reverse Phase (RP) Chromatography is very Versatile and

Dynamic

About 80-90% of the compounds are analyzed by RP-HPLC

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Normal Phase Vs Reverse Phase Chromatography

Normal Phase Reversed Phase

1. Stationary phase Polar (silica gel) Non-polar (Silica derivative
with C8 or C18)
Non-polar Polar
2. Mobile phase
(organic solvents, e.g. (Aqueous/organic solvents
Hexane, Cyclohexane, like Water, Methanol,
etc.) Acetonitrile, IPA, etc.)
3. Sample Non-polar elutes fastest Polar elutes fastest
movement

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 Reverse Phase Synthesis/Preparation

Silica Gel (Base Material) Derivatized Silica Gel (End capping)

O O O O O O
| | | | | |
OSiOSiOSiOH OSiOSiOSiOR Where R = C8H17 or C18H37
| | | | | | Hydrocarbon chain
O O O O O O (i.e. Octyl C8, or Octadecyl,
| | | | | | C18 derivative)
OSiOSiOSiOH OSiOSiOSiOR
| | | | | |
O O O O O O

Bulk (SiO2)x surface Bulk (SiO2)x surface

Relatively polar surface Relatively Non-polar surface

“Normal phase” “Reverse phase”
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 Mobile Phase for Reverse phase HPLC

Mixture of Water and Organic solvents are used.

• Water:
“Ultrapure water” should be used (i.e. filtered through Millipore Filtration system).
- Commercially “distilled water for HPLC” is also available. (but it is costly).

• Organic Solvents
– HPLC-grade solvents should be used (Extra pure/costlier than synthesis grade
solvents), e.g. Methanol, Acetonitrile, THF, DMSO

– 100% Water should never be used in Reverse phase chromatography

(Otherwise hydrophobic collapse of brush-like structure of C18 chains, i.e. dewetting occurs,
Water repels the hydrophobic / Non-polar stationary phase, C18)
Organic modifiers like MeOH or AcN need to be added.. At least 2%

– Aqueous Buffers are used with some % of organic modifiers for acids and bases
(e.g. acidic buffer like Sodium or ammonium dihydrogen phosphate with pH 4 is
used to suppress the ionization of carboxylic acids to avoid peak tailing problem).
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 Mixing, Filtration, and Offline Degassing of the Eluent

Decompression Membrane filter with pore

by aspirator size of 0.20 µm

Decompression
by aspirator

Ultrasonic
Machine

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 HPLC Instrumentation

Schematic HPLC System Real Agilent HPLC System

UV-Visible detector
Solvent Reservoirs
Controller

Solvent Cabinet Vacuum Degasser

Binary Pump

Autosampler

Thermostatted
Column Compartment

Detector

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 HPLC Configuration
Manual Injection

Load - Inject
1. Pump: (Transports mobile phase through system)
 Isocratic elution
 Gradient elution

2. Sample Injection: (Injects sample onto the column)

 Manual Injection
 Automated injection Sample Loop

3. Column oven compartment:

 Maintains constant temperature (Rt reproducibility)
 Temp. variations especially in summer

4. Detectors: (Identification & Quantification of peaks) Inject

 UV-Visible detector (Versatile and Sensitive detector)
 Refractive index (RI) detector
 Evaporative light scattering detector (ELSD)
 MS detector

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 UV-VIS Absorbance Detector Principle (Beer-Lambert law)

C: Concentration
Detection cell

I0 I

A
b
C
A = e·C·b = –log (I / I0)
(A : absorbance, e: absorption coefficient, C = concentration of sample,
b = path length, I = Intensity of light after passing through sample, I0 = Initial
intensity of light)
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 Making HPLC System ready for Analysis
1. Purging Mobile Phase
2. Setting Mobile phase composition/Solvent Bottles A, B, C, D
(Premixed and Online mixing)
3. Setting Column flow rate (typically 0.5 -1.0 mL/min)
4. Column oven temperature (Typically 40 0C)
5. Selecting Detection Wavelength (λmax)
6. Sample injection (Sample Volumes: 5 – 40 μL)
7. Optimizing Elution time or chromatographic run time

Execution of HPLC Run

1. HPLC Column conditioning (Noise/Drift/Stabilization of Baseline)
2. Preparing Sample list sequence/Data file name/sample name
3. Run start
4. Monitoring Chromatogram/Zoom/X-Y Scale alteration
5. Data Acquisition/Peak Integration
6. Viewing and Printing Results/Reports
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Method Validation :
To evaluate the Reliability of Analytical results

Purpose
After an analytical method is developed, it should be validated
to ensure that it is useful for its intended purpose.

Method validation parameters

1. Specificity/Selectivity
2. Precision (Repeatability of retention times and Peak areas)
3. Accuracy (True value Vs Measured value)
4. Linearity/System calibration
5. Range
6. Limit of detection (LOD)
7. Limit of quantification (LOQ)
8. Method Ruggedness

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 Accuracy & Precision
True value

Accurate but Inaccurate but

imprecise precise

Inaccurate &
imprecise Accurate and precise

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 Linearity studies /Calibration Curve
y/x = m, Slope i.e. Response factor
y = mx + C
Concentration Peak Area (Zero intercept, c= 0, curve passing through origin)

A1 Calibration curve
C1
A4

A2 A3

Peak area
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
Concentration 24
 Noise, LOD and LOQ

S/N = 10 Noise: Random fluctuation of

an electronic signal

Limit of detection/LOD: An
amount or conc. of a sample
that produces the peak area
S/N = 3 three times the noise value
(S/N = 3)
Noise
Limit of quantification/LOQ: An
amount or conc. of a sample
that produces the peak area
ten times the noise value
(S/N = 10)

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 Maintenance/Troubleshooting of HPLC System
Sr. Care to be taken Consequences
No.

1 Always use MilliQ Water and solvents filtered through 0.2 μm filter for Clogging of column, high pressure, column
preparing mobile phases damage

2 Always degas mobile phase to expel out dissolved air. Causes erratic column flow and poor peak
shapes.
3 Always ensure that there is sufficient solvents available in the reservoir. Entry of air in the system .

4 Always prime pump (Purging) before every analysis and periodically Stagnant mobile phase in reservoir and
even when system is not in use tubing lines - may cause bacterial
growth
5 The detector should be having a good lamp in it (Periodical Checking) Low light intensity interfering absorbance,
hence low sensitivity of detector affecting
LOD and LOQ values
6 The HPLC columns shouldn’t be run at high pressures (A typical column Column bed disturbance/ de-packing/Peak
flow ranges from 0.5 to 1.0 mL/min). shape distortion

7 Don’t overload column with concentrated samples, avoid injecting large Peak distortion problems, Peak tailing or
volume samples, typical injections 5 – 40 μL fronting problems/Low resolution
8 After HPLC run is over, column should be washed with 50:50, Water: Enhances life of column and performance
MeOH or 50:50, Water: Acetonitrile and stored in a solvent as per
storage conditions specified by a column manufacturer (generally
recommended to keep it in 100% Acetonitrile)

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27
 Strong ion exchangers bear functional groups [e.g., quaternary amines or sulfonic acids]
that are always ionized. They are typically used to retain and separate weak ions. These
weak ions may be eluted by displacement with a mobile phase containing ions that are
more strongly attracted to the stationary phase sites. Alternately, weak ions may be
retained on the column, then neutralized by in situ changing the pH of the mobile phase,
causing them to lose their attraction and elute.
Weak ion exchangers [e.g., with secondary-amine or carboxylic-acid functions] may be
neutralized above or below a certain pH value and lose their ability to retain ions by charge.
When charged, they are used to retain and separate strong ions. If these ions cannot be
eluted by displacement, then the stationary phase exchange sites may be neutralized,
shutting off the ionic attraction, and permitting elution of the charged analytes.
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Some common Types of HPLC
1. Ion-exchange chromatography : For Ionic samples (The stationary bed has an ionically
charged surface of opposite charge to the sample ions. The mobile phase is an aqueous
buffer, where both pH and ionic strength are used to control elution time.

2. Size exclusion chromatography (or Gel Permeation Chromatography, GPC):

The column is filled with material having precisely controlled pore sizes. Larger
molecules are rapidly moved through the column and the smaller molecules penetrate
inside the porous of the packing particles and elute later.
(Useful for analysis of polymers)

3. Normal phase chromatography: Polar stationary phase & non-polar mobile phase.

4. Reverse Phase chromatography: Non-Polar stationary phase & polar mobile phase

About 80-90% of the compounds are analyzed by Reverse

Phase (RP) Chromatography/ Very dynamic and versatile.
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