ZZ1-HPLC 2018-Adsn PDF

H : High
P : Performance (Pressure)
L : Liquid
C : Chromatography
ATEP DIAN SUPARDAN
What is HPLC used for ?
1. Separation of mixed components
2. Qualitative analysis / Quantitative analysis
3. Preparation of interest components
Separation analysis and/or preparation

of interest components
GC : Gas chromatography
TLC : Thin layer chromatography
IC : Ion chromatography
Separation and Analysis
Qualitative analysis
What are components A, B and C ?
Quantitative analysis
What is the concentration of
components A, B and C ?
Results obtained by HPLC
Chromatogram containing three peaks

Qualitative analysis (identification) and
Quantitative analysis (determination)
Can be performed using the information contained in the
chromatogram
Chromatography : Method
Chromatogram : Results
Chromatograph : Instrument
Why high pressure?
In HPLC the stationary phase has two characters :
- Has small particles size (5- 10 µm).
- And packed under high pressure.
Reduction of the particle size of the stationary phase

leads to:
- Leaving less space for the mobile phase to pass
through.
- Decrease the flow rate of the liquid mobile phase.
- Thus pressure from 1000 to 5000 psi, pound per

square inch (68 to 340 atm.) is applied to overcome the
obstructive effect of the fine particles.
Chromatogram
Identification
What is component A?
Component A elutes the same time as a caffeine peak.
Component A is identified as caffeine.

Determination
What is the concentration of component A?
Peak area (or height) is proportional to the concentration

(or amount) of the component.
The concentration of component A(caffeine) is determined by

comparing the peak area with that of the standard caffeine peak.
Separation Mechanism
Separation is determined by column (packing material) and mobile phase (solvent).
Mobile phase elutes components.

Packing materials retain components in the column.
You’ve Got a Problem to Solve
I need a quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.
I’ll need a separation

technique.
Separation Techniques
I have two separation techniques in my lab,

High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
How can We Analyze the Sample?
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose 5
2
3
Zorbax NH2 (4.6 x 250 mm) mAU
4
70/30 Acetonitrile/Water 1
6
1 mL/min Detect=Refractive Index
time
Five modes in HPLC
Packing
LC mode Mobile phase Interaction
materials
Normal phase Adsorption

Silica gel n-Hexane/IPE
chromatography
Reversed phase Silica-

MeOH/Water Hydrophobic
chromatography C18(ODS)
Size exclusion Porous

THF Gel permeation
chromatography polymer
Ion exchange Ion exchange

Buffer sol. Ion exchange
chromatography gel
Affinity Packings with

Buffer sol. Affinity
chromatography ligand
Modes of High Performance Liquid Chromatography
Separation Instrumentation/Machine Examples
• High Performance Liquid Chromatography (HPLC)
• size exclusion (SEC)
• ion exchange (IEX)
• ion exclusion (organic acid, mono- and oligosaccharide)
• reverse phase (RP)
• normal phase (NP)
• hydrophobic interaction chromatography (HIC)
• hydrophilic interaction chromatography (HILIC)
• etc…
• Ion Chromatography (IC)
• ions: cations, inorganic anions, organic anions
• (e.g. organic acids, phytic acid)
• carbohydrates
• Capillary Electrophoresis (CE)
• ions: cations, inorganic anions, organic anions
• proteins: MW, pI, glycosylation, post-translation modifications
• carbohydrates: DP1-DP40+
http://www.waters.com/waters/nav.htm?cid=10048919
November 1, 2010
Advantages to HPLC
• Higher resolution and speed of analysis
• Rapid and precise quantitative analysis
• Typical analysis time of 5-20 min, precision <0.5-1% RSD
• Automated analysis
• Using autosampler and data system for unattended analysis and report generation
• High sensitivity detection
• Detection limits of ng to pg
• Quantitative sample recovery
• Preparative technique from mg to kg quantities
• Amenable to diverse samples
• Can handle >60% of all existing compounds vs. 15% for GC
• Can analyze samples with little or minimal preparation
• Greater reproducibility due to close control of the
parameters affecting the efficiency of separation
• Advantages of HPLC are result of 2 major advances:
• stationary supports with very small particle sizes and large surface
areas
• appliance of high pressure to solvent flow
HPLC Applications
Typical Applications of HPLC Chromatography
HPLC Chromatography
1. Pump System. Mobil phase pressures up to 6000 psi are
necessary to achieve reasonable column elution times (~
minutes). Typical flow rates are 0.1 to 10 mL/minute.
2. Injection System. Used to introduce small samples (0.1 to 500
µL) into the carrier stream under high pressure.
3. Reservoirs (Solvents). Multiple solvents are necessary for
performing gradient elution's (i.e. changing the polarity of the
mobil phase during a run).
4. Chromatographic Column. Typically 10-30 cm in length
containing a packing of 5-10 µm diameter. Many types of
columns are available, depending on the type of liquid
chromatography desired.
5. Detector. Many types are available including UV, IR, refractive
index, fluorescence, conductivity, mass spectrometry, and
electrochemical. Diode array detectors are used when
wavelength scans are desired.
Schematic of an HPLC System
HPLC Basic Instrumentation
Liquid phase samples (mixtures) are injected onto an LC column usually using a syringe and
specially devised injection valve. The sample is swept onto the chromatographic column by
the flowing mobile phase and chromatographic separation occurs as the mixture travels
down the column. Normal HPLC detectors detect the elution of a compound from the end
of the column based on some physical characteristic such as ultraviolet light absorption,
ability to fluoresce, or the difference in index of refraction between the analyte and the
mobile phase itself.
HPLC Instrumentation
The HPLC Machine
autosampler
differential
+ heater refractive index
27
Daniel C. Harris, Quantitative Chemical Analysis, 7 th ed., W.H. Freeman and Co., NY, 2007.
HPLC Analysis Parameters
Hewlett-Packard Series 1100 HPLC
HPLC Column
HPLC Pump
HPLC Autosampler and Injector
HPLC Detector - UV/Visible Spectrophotometer
HPLC Waste Collection
Agilent 1200 Series purification system Analytical scale (AS)
Agilent’s approach - dedicated systems
• Purification of sample amounts

up to the low mg range
• for flow rates below 5 (10)
mL/min
• Minimum delay volumes for
high separation performance
resulting in high recovery and
purity
Agilent 1200 Series purification system Preparative scale (PS)
Agilent’s approach - dedicated systems
•Purification for sample amounts < 1 g

•Flow rates up to 100 mL/min
Some Quick HPLC Maintenance and Troubleshooting Tips
• Preventative Maintenance: only recommendations?
– guard column or cartridge: replace routinely
– routine cleaning of main tubing: 2/year
• disconnect column…10% HNO3 and be very careful!
– purge reference cell of differential refractive index detector: every bag change
– do not allow mobile phase reservoirs to become empty: replace pump seals
– replace pump seals: 1/year or whenever system was run dry
– clean (solvent sonication) or replace check valves: 1/year or whenever system
was run dry
– replace in-line porous frit filters (MP reservoirs, pumps, autosamplers, and
even some detectors)
– the right i.d. tubing…the proper tubing cuts
– remove sample particulates
– clean laboratory environment (e.g. remove dust from instrument exhaust
fans)
• Troubleshooting
– know your system’s vital signs…i.e. healthy specs (pressure and real flow rate)
– for tracking down clogs…i.e. high pressure…always disconnect at back and
work forward
– column regeneration
38
• broad peaks…need to replenish co-ion
(mobile phase)
HPLC solvents.
• Generally we want a significant difference between the
polarities of the s.p. and the m.p.(partitioning)  K = Cs/Cm
• Almost all reversed phase separations (polar m.p. &
nonpolar s.p.) can be carried out with combination of
acetonitrile (CH3CN), and/or methanol, and water as a m.p.
• Separation of most organic compounds can be handled by
C-18 stationary phases.
• Most mobile compositions can be handled by

either CH3CN/H2O or CH3OH/H2O
– Solvents must be miscible e.g. water/ethanol. An

immiscible solvent system such as water/toluene would
create a mess in the column!
Reservoir for solvents (mobile phase)
-Mobile phase is usually organic or
aqueous or mixture of both.
- Mobile phase is placed in bottles of
glass.
• 1- Pure
• 2 - Low viscosity
• 3-Chemically inert
• 4- Low price
• 5- Compatible with detector
• 6- Solubility of the sample
Miscible with water, such as

acetonitrile, methanol, or isopropanol.
Treatment of mobile phase
A- Filtration before entering the column.
B- Degassing using degasser.

1- Heating with stirring
2- Applying vacuum,
3- Passing nitrogen or helium
4- Ultrasound
C- Pre-saturation with the stationary phase in case

of liquid liquid chromatography.
Solvent Filters
Precolumn Filter
• Used between the injector and
Solvent Inlet Filer guard column.
• Stainless Steel or • 2 to 0.5 micron
glass with 10 micron • Removes particulates from
porosity. sample
• Removes particulates and autosampler wear debris.
from solvent. • Must be well designed to
prevent
dispersion.
HPLC Degassing
• Degassing removes
dissolved air that
interferes with check valve
operation
• Helium sparge
– Gas line from the tank
directly in the solvent bottle
• Vacuum degassing
– Sonicate before connecting
to the system
– Online with a degassing unit
Mobile phase selection
Solvent strength (o ) is a quantitative representation of solvent strength.
For non-electrolytic solvents, solvent strength is synonymous with “polarity” in
normal phase LC. Strong or polar solvents are characterized by their ability to
dissolve polar samples and to elute solutes with low capacity factors(k’) in
normal phase LC. In reversed phase LC there is an inverse relationship between
solvent strength and chromatographic elution power.
For example, water is a strong solvent but has little elution power in RP-LC and
must, therefore, be mixed with a less polar organic midifier to elute a solute
with a low k’ value.
The eluotropic series gives the chromatographer a method of influencing the k’
values of sample components.
Decreasing the solvent strength, k’ values may be increased. To increase
chromatographic resolution per unit time during the course of a
chromatographic run, the solvent strength can be increased in a stepwise or
continuous manner causing k’ values to change. This is known as solvent
programming.
Properties of some common solvents used in HPLC
Solvent UV cut-off Refractiv Boilin Viscosity Solvent Solvent Selectivity
(nm) e Index g Point (cP, 25 C) Polarity Strength Group
(C) Parameter(P’) Parameter()
Isooctane 197 1.389 99 0.47 0.1 0.01 -
N-Hexane 190 1.372 69 0.30 0.1 0.01 -
Methyl t-butyl 210 1.369 56 0.27 2.5 0.35 Ⅰ
ether
Benzene 278 1.501 81 0.65 2.7 0.32 Ⅶ
Methylene 233 1.421 40 0.41 3.1 0.42 Ⅴ
chloride
n-Propanol 240 1.385 97 1.9 4.0 0.82 Ⅱ
Tetrahydrofuran 212 1.405 66 0.46 4.0 0.82 Ⅱ
Ethyl acetate 256 1.370 77 0.43 4.4 0.58 Ⅵa
Chloroform 245 1.443 61 0.53 4.1 0.40 Ⅷ
Dioxane 215 1.420 101 1.2 4.8 0.56 Ⅵa
Acetone 330 1.356 56 0.3 5.1 0.56 Ⅵa
Ethanol 210 1.359 78 1.08 4.3 0.88 Ⅱ
Acetic acid 1.370 118 1.1 6.0 Large Ⅳ
Acetonitrile 190 1.341 82 0.34 5.8 0.65 Ⅵa
Methanol 205 1.326 65 0.54 5.1 0.95 Ⅱ
Water 1.333 100 0.89 10.2 very large Ⅷ
ST = i Siqi
ST = total solvent strength of the mixture
Si = solvent strength weighting factor
qi= volume fraction of solvent in the mixture
Example) methanol/water ( 60 : 40 v/v %)

ST = i Siqi = SCH3OHqCH3OH + SH2OqH2O
ST = (2.6)(0.6) + (0)(0.4) = 1.56
iso-eluotropic solvents
Binary system
acetonitrile/water
1.56 = (3.2)(qCH3CN) (0)(qH2O)
(qCH3CN) = 0.49 = 49 %
Ternary system
methanol / tetrahydrofuran / water (35:10:55)
PH of mobile phase
The pH of the solvent (water) may be adjusted using

phosphate or perchlorate or trifloroacetate acid or sulphate
buffer.
The selectivity of HPLC is affected by :

1- Type of mobile phase, organic or aqueous.
2- The composition of the mobile phase, whether one solvent
or more.
3- The pH of the mobile phase.
• Affects ionizable compounds
– organic acids
– organic bases
• In reversed phase we need to suppress ionization as
much as possible
• May need very precise pH control
Effect of buffer used in separation of xanthene alkaloids
• TFA
• Incomplete separation
• HClO4
• No separation for 2 & 3
• H2SO4
• Best separation
Mobile Phase Composition Effect on Selectivity
30% MeCN 45% MeOH

70% Water 55% Water
Fast Slow and better separation
Methanol and water give slow and better separation while use of actonitrile
and water give fast and bad separation
30x0.46 cm C-18, 1.5 mL.min, 254 nm, 10 mg each. Snyder and Kirkland,
introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287.
Effect of Solvent
• Water is “weak” solvent

• Increased organic ---> decreased retention
• Organic must be miscible with water
% of Mobile phase B (MeOH) and separation selectivity
Low % of B gives
High % of B gives slow and slightly better
fast and bad separation separation
pH Effect on Retention
1. Salicylic acid
2. Phenobarbitone
3. Phenacetin
4. Nicotine
5. Methylampohetamine
30x0.4 cm C-18, 10 mm, 2

mL/min, UV 220 nm
Snyder and Kirkland, Introduction to Modern

Liquid Chromatography, Wiley, 1979, p. 288.
Use of Buffers
• 0.1 pH unit ---> significant effect on retention
• Buffer mobile phase for pH reproducibility
• pH of buffer should be within 1 pH unit of pKa
of acid (best at pH = pKa)
• Buffers weak (100 mM or less)
• Check solubility
Silica Surface
Silanol Activity
• RP ligands occupy
about 50% of
silanols
• Others are “active”
• Weak acids
Dealing with Residual Silanols
• Silanols cause peak tailing and
excessive retention
• Endcapping
– bond a smaller group
(helps a little)
• Pre-treatment of silica
Silanol Interactions
– fully hydroxylated best
• Hydrogen bonding
– high purity best
• Dipole-dipole
• Ion exchange
• Low pH --> silanols protonated
• Add basic modifier (TEA) to
compete for sties
pH Effect on Tailing
RP Optimization
RP Optimization
4/18/2018 Chem 253 - Chapter 25 63
Liquid Chromatography
Instrumentation – Mobile Phase Delivery
Less retention
• Example of solvent changes OH OH
to affect selectivity:
H3C CH3 CH3
O
O O
More
– RP-HPLC Separation of syringols
retention
from guaiacols R R
– Difference is in 2nd MeOH Syringols Guaiacols

group
– Water/Acetonitrile eluents
produce poor syringol/guaiacol HPLC-UV
HPLC Sample
Sample11(MeOH/0.1%TFA)
(ACN/0.1%TFA) acetovanillone
acetosyringone
separation factors 350
350
acetosyringone
acetovanillone
cinnamic
cinnamicacid
acid
300
300 isoeugenol
isoeugenol
– Water/Methanol works better 250
250
Absorbance
Absorbance
200
200
syringic
syringicacid
acid
(although greater retention 150

150
100
100
with MeOH of syringol is 5050

00
-50
-50
counter intuitive) 00 55 1010 1515 20 20
Time
Time (minutes)
(minutes)
• Optimization of Mobile
Phase Composition Acetonitrile
– Separation should be (40% in water)
perfomed on three different
water/organic systems
20% ACN, 25%
– Then additional separations MeOH, water
can be carried out using 3
component mobile phases
THF (30% in
– Patterns in retention can be Methanol (50% water)
used to optimize mobile in water)
phase composition
• Mobile Phase Selection – pH
Buffering O O
– In reversed-phase HPLC,
solute generally must be non- OH O
-
ionized to be retained
– pH is adjusted by adding
buffer in water/organic retained unretained
modifier
– pH at pKa means retention
factor about half of non- NHNH
2 3
+
-
ionized acid retention time O
O
– In ion-exchange S
chromatography, pH should be O CH3
in range needed to produce

ions
– In ion-pairing RP-HPLC, an Benzyl amine Ion pair reagent = pentane
(conj.
ion-pairing reagent is added acid pKa = 9.35)
sulfonic acid (sodium salt)
Non-ionized only at
high pH
• Solvent Flow
– HPLC requires high pressures and thus
specific pumps
– The solvent also needs low levels of dissolved
gases for pumps to function
– For the simplest “dedicated” HPLC, a single
solvent reservoir and pump is needed
– For gradients and/or more method
development work, switching between
different solvents is needed
HPLC – Effect of Modifier
Acetonitrile : Water
60:40 50:50 40:60

Viscosity vs. Pressure
The higher the solvent viscosity, the harder it is for the

solvent to move through a column, and the more pressure in
required to move the solvent at a specific velocity. The pressure
required to move a solvent through a column can be estimated by
the following formula:
P = 250 L  F / Dp Dc 2 2
Where P = pressure drop in psi. F = flow rate (mL/min)

L = column length (cm) Dp = particle diameter (mm)
= solvent viscosity (cP) Dc = column diameter (cm)
EXAMPLE
P = 250 L  F / Dp2 Dc2
column length = 15 cm, column diameter = 0.5 cm,
particle diameter = 5 mm, flowrate = 2.0 mL/min
For water n = 1.0

= 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psi
For methanol n = 0.54

= 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psi
For 60% water n = 1.9

=250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi
Gradient elution method
Elution Techniques (Programinig)
Gradient elution:
The mobile phase composition
is changed during the
separation process.
Gradient elution is divided

into two types:
A- Continuous (linear)
B- Discontinuous (stepwise)
Isocratic elution:
The mobile phase composition
remains constant throughout
the separation procedure.
Elution Techniques (Programinig)
Isocratic Separation
• Same mobile phase concentration throughout the separation
• Use 1 pump and pre-mix solvents
• Use 1 pump and a valve for 4 different solvents
• Use 2 pumps and vary the amount coming from each pump
• 1 pump and premixing
• 4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water
• 1 pump with valve and premixing
To Column
ABCD
A = 80% Methanol, 20% Water

B = 70% Methanol, 30% Water
C = 60% Methanol, 40% Water
D = 50% Methanol, 50% Water
• 1 pump with mixer – let the pump do the work!
To Column
ABCD
Method 1: A.CONC = 20%, B.CONC = 80%

Low Pressure Gradient
• 1 Pump, solvents are mixed before the pump
To Column
ABCD
• REQUIRES degassing
Advantages of gradient elution technique
1- Shortening the time of analysis.
2- Reduces tailing, gives sharp peak.
3- Increases the sensitivity of analysis.
4- Decreases the retention of the later-
eluting components so that they elute
faster.
HPLC System Types
• High Pressure Gradient
– Multiple pumps are used with a mixer after the
pumps
• Low Pressure Gradient

– Solvents are mixed before the pump
High Pressure Gradient
• Binary Gradient  Ternary Gradient
• 2 Pumps and Mixer  3 Pumps and Mixer
……….
……….
……….
………. ……….
……….
……….
……….
……….
………. ……….
……….
……….
……….
……….
Low Pressure Gradient
• 1 Pump, solvents are mixed before the pump
To Column
ABCD
• REQUIRES degassing
4. Gradient elution method
For separation of a sample containing many components
MeOH(100)
Gradient
Mobile phase
MeOH/Water 0.5M
(50/50)
0.1M KH2PO4
Step wise
0.01M
0 5 10 15 20 25
Time(min)
Advantage of gradient elution method
Isocratic elution method Gradient elution method
A
A
Finepak SIL C18

B
MeOH/1%
AcOH(40/60)
B
*
*
A MeOH/1% AcOH
MeOH/1% AcOH(30/70) 30/70→45/55
A :Linear Gradient、16min
Chlorogenic acid
B : Rutin
B
* : Impurity
Precautions in gradient elution method
- Can the gradient save time ?

- Reproducibility
- Baseline
- Ghost peak
- Salt
Effect of temperature on retention time

3
1 2 4 Finepak SIL C18
60*C
CH3CN/H2O(90/10)
Sample：
1. Benzene
2. Anthracene
3. Pyrene
2 4. Benz(a)pyrene
40 *C 1 3
4
1 2
20 *C
3
4
0 2 4 6 8 10 12 (min)
Chromatograms illustrating separations of mixtures of ionic and nonionic compounds by ion
pair chromatography
Pumping system.
The pumping system must...

1) Generate pressures up to ~6,000 psi
2) Give a pulse-free output (via a pulse damper).
3) Give flow rates of ~0.1 - 10 mL/minute.
4) Give flow reproducibility of 0.5% or better.
5) Resist corrosion by a variety of solvents.
6) Corrosion resistance
Two groups of pumps:

1)Constant pressure
2)Constant volume
Three types of pumps are available:

1)Reciprocating pumps (90% of Commercial HPLC; produce pulse flow)
2)Displacement pumps (produce flow that are independent of viscosity
and back pressure)
3) Pneumatic pumps (cannot do gradient and pressure less than 2000psi)
Functions of the Solvent
Delivery System
The solvent delivery system

has three basic functions:
1. Provide accurate and
constant flow.
2. Provide accurate mobile
phase compositions.
3. Provide the force
necessary to push the
mobile phase through the
tightly packed column.
Multichannel Gradient Valve
• Determines mobile phase
composition.
• Largest solvent plug fills first.
• Agilent 1100 and 1200
quaternary pump.
Frits and Filters
Purge valve
PTFE Frit
Frits, Filters, and Sieves are used to protect other parts of the LC from pump and seal material.
Damping Units
Damping
Unit
Pump
Ripple
2%
P/P
Pressure
• Filled with compressible liquid separated

from the mobile phase by a membrane.
• Pressure ripples reduced to < 2% original
value.
95
Gradient Formation
Low Pressure Gradient High Pressure Gradient

HPLC Pumps: Reciprocating Piston Pumps
consist of a small motor driven piston
which moves rapidly back and forth in a
hydraulic chamber that may vary from 35-
400 µL in volume. On the back stroke, the
separation column valve is closed, and the
piston pulls in solvent from the mobile
phase reservoir. On the forward stroke,
the pump pushes solvent out to the
column from the reservoir. A wide range
of flow rates can be attained by altering
the piston stroke volume during each
cycle, or by altering the stroke frequency.
Dual and triple head pumps consist of
identical piston-chamber units which
operate at 180 or 120 degrees out of
phase. This type of pump system is
significantly smoother because one pump
is filling while the other is in the delivery
cycle.
Reciprocating pump
Ballvalves for Reciprocating Piston Pumps
Gold Seal
Sapphire
Insert
Ruby Ball
Spring
Insert
Dual Piston Pump
Two pistons with

equal volume (10 µL
each)
During each stroke, 10
µL is delivered
Best for low flow rates
(< 1 mL/min)
Little to NO pulsation,
so it’s ideal for pulse
sensitive detectors
like RID and CDD
High-pressure piston pump for HPLC. Solvent at the left passes through an electronic valve synchronized
with the large piston and designed to minimize the formation of solvent vapor bubbles during the intake
stroke. The spring-loaded outlet valve maintains a constant outlet pressure, and the damper further reduces
pressure surge. Pressure surge from the first piston are decreased in the damper that “breathes” against a
constant outside pressure, Pressure surge are typically <1% of the operating pressure. As the large piston
draws in liquid, the small piston propels liquid to the column. During the return stroke of the small piston,
the large piston delivers solvent into the expanding chamber of the small piston. Part of the solvent fills
the chamber, while the remainder flows to the column. Delivery rate is controlled by the stroke
volumes.[courtesy Hewlett-Packard Co., Palo Alto, CA.]
Dual Piston Parallel Pump
Check
Valves
Rotary
Switching
Valve
Pumphead
Piston
A B
Single Combined
Piston Delivery
Delivery
Piston 'A' Advancing Piston B Retracting

Dual Piston in Series Pump
 First piston displaces

solvent at twice the
speed and stroke volume
of the second piston.
 Provides constant flow and the

pressure necessary to get
through column.
103
HPLC Pumps: Syringe Type Pumps
are most suitable for small bore columns because this pump delivers only a finite
volume of mobile phase before it has to be refilled. These pumps have a volume
between 250 to 500 mL. The pump operates by a motorized lead screw that delivers
mobile phase to the column at a constant rate. The rate of solvent delivery is
controlled by changing the voltage on the motor.
• Pulse-free
output,
limited mobile
phase
capacity.
HPLC Pumps
In Constant Pressure Pumps the mobile phase is driven through the column with
the use of pressure from a gas cylinder. A low-pressure gas source is needed to
generate high liquid pressures. The valving arrangement allows the rapid refill of the
solvent chamber whose capacity is about 70 mL. This provides continuous mobile
phase flow rates.
Valves Used With Pumps
• Solvent Selection – 2 Solvents Per Pump
– Use for ……….
solvent switching
……….
……….
……….
……….
……….
1 2 1 2 1 2
……….
……….
……….
A
A B C
– Use for pump loading of large sample volumes
Pump B – strong gradient solvent. Form the gradient with B.CONC
command
Pump A – weak gradient solvent and sample loading
12 Weak
gradient Sample
solvent
A B C
– Use for low pressure gradient formation
To Column
Combine any proportion of A/B/C/D.

ABCD
REQUIRES additional mixing before the injector.
– Use for different gradients in method development
Pump A Pump B
A A
B
B B
C C
D D Pump A Pump B
A
A A, B, C, D
4 Pairs B
B A, B, C, D
C A, B, C, D
A D A, B, C, D
16 Combinations
Pump Troubleshooting
• No pressure, or fluctuating pressure
– Pump may not be completely full of liquid – check
solvent inlet line
– Air in check valve – always degas mobile phase!
– “Stuck” check valve – the pump may have been idle
for too long and solvent has dried inside the check
valve.
Poor quality solvent: may contain resins that coat
the ball inside the check valve, and that film won’t
let the ball seat properly
• High Pressure
– Outlet frit may be blocked with particles from
mobile phase or seal material
Pump Troubleshooting
• Leaks
– Damage to seal and/or plunger due to several
factors
• Misaligned plunger
• Solvent incompatibility with seal material
• Salt crystal buildup from buffers – use a rinse kit!
• Retention Time Reproducibility

– For a dual piston pump, only one side may be filled
with liquid – check solvent inlet lines
– Temperature change (may not be the pump’s fault)
• A 1o shift in temperature can result in a 1-2% shift in
retention time
• Avoid drafty locations in the lab
• Use a column oven when possible
Chromatography – Dead Volume Mixing
SAMPLE INJECTOR
A manual sample injector that is typically used comprises of
a 6-port 2-position valve that includes a 10 or 500 µl fixed
sample loop. In one configuration, the flow from the pump
is sent directly into the column; when the position is
switched the flow from the pump is diverted via the sample
loop into the column, thereby performing a sample injection.
Valves with electrically or pneumatically actuated position
switches are also commercially available.
Automated sample injectors (autosamplers) that can store
and sequentially inject multiple samples are popular in
quality control applications and for high-throughput
screening.
Reproducible introduction of the sample volume into the
mobile phase flow.
Two major designs:

Automatic Injectors or Manual Injectors
(Injection port)
1- Manual injection 2-Automated injection

Rheodyne manual sample
injector and sample loops (ss or
PEEK)
Scale Sample Volume
Micro 2 µL to 500 µL
Analytical 1.0 µL to 5.0 mL
Preparative 100 µL to 10 mL
http://www.rheodyne.com/sampinj.html
Manual Injectors
Load - Inject
Front View Inject
Rear View
Manual Injectors
Sample Injection – Manual
• Manual Injector with Syringe
– Fixed loop of varying sizes (1 to 20 mL or more)
– Fill with syringes of varying sizes
– Can include a switch to start a data syste
Picture from http://www.rheodyne.com/products/fluidic/manualapps/manualsample.asp#

Sistem injeksi simpal
Manual Injectors
Sample Load
From Pump
Solvent in
Solvent out Sample in
To column
From Pump
Solvent in
Solvent out Sample in
To column
Sample Inject
122
Sample Injection – Automatic
• Fixed-Loop Autosampler
– Loop is installed on the valve and can be changed
for different injection volumes
– External syringe draws sample and fills loop
• Advantages: low cost, rugged, few moving

parts
• Disadvantages: Poor performance for low
volume injections, higher carryover, always
some sample loss
Automatic Injectors
Step 1 Step 2
Step 3
HPLC Sample Injection Loop
The injection loop is a critical component of an HPLC and is potentially one
of the largest sources of excess HPLC system volume. The sample is
injected into the loop while the loop is switched out of the HPLC flow
path. After the loop is filled with sample it is switched back into the flow
path and the sample is swept onto the head of the HPLC column for later
elution or the sample is injected directly into a mass spectrometer as part of
a flow injection analysis.
PEEK™ Loop
Note:PEEK™ (polyetheretherketone) is a
registered trade mark of Victrex plc.
Sample Injection – Fixed Loop
• External syringe draws sample, then fills the

fixed-volume loop attached to the valve.
Sample Injection – Automatic
• Needle-in-the-flowpath autosampler
– Sample loop and needle are a single piece of
tubing
– Loop and needle are cleaned during the run
– Metering pump draws sample very precisely
• Advantages: no sample loss, low carryover

• Disadvantages: higher cost, more delay
volume for gradient
Sample Injection to Flow Path
• Sample Loading
 Sample Injection –
Everything drawn
into the needle goes
to the column.
Rinsing After Injection
• Rinse liquid flows
through ports 5 and
6 of the high
pressure valve.
 Sample aspiration uses

port 5.
 If air is present around
port 5, injection
reproducibility will be
low.
 Rinse liquid MUST be
degassed!
Rotor Seals
Rotor Seal
found within
valve
Autosampler Troubleshooting
• High Pressure
– Particulates from mobile phase, sample, pump
may be trapped in the inlet tubing or valve
• Filter mobile phase AND sample when possible
• Leaks
– Fittings may be loose on the valve
• Tighten fittings properly and don’t exceed the pressure
limit of the autosampler
Autosampler Troubleshooting
• Area % Reproducibility
– Always degas rinse phase, and use some volume
of liquid for rinsing to keep all flow paths in the
valves full of liquid
– Make sure the needle stroke is deep enough to
draw sample from the vial
– Check for leaks on the valve fittings, and the
connection to the column inlet
Columns
a. Columns are usually stainless steel tubes.
b. Lengths are typically 10 - 30
centimeters.
c. Inside diameters of 4 - 40 mm are
typical; newer high-performance columns
are 1 - 4.6 mm.
d. Particulate packing size is typically 5 -
10 micrometers.
HPLC column with replaceable

guard column to collect
irreversibly adsorbed impurities.
Titanium frits distribute the
liquid evenly over the diameter
of the column.
Column dimensions
–Length: balance between Diameter
Typical
flow, pressure and Type
(mm)
Flow Rate
(mL/min)
efficiency Preparativ >7.8 >3
e
–Diameter:
Analytical 4.6 1
• Choice depends on separation
purpose Microbore <1 < 0.05
• Preparative for isolation of larger

quantities
• Microbore usually results in
smaller mass detection limits but
greater concentration detection
limits
• Special care is needed using
microbore with sample injection,
pump stability, and extra-column
broadening
Precolumn filters
- 2 types porous stainless frit 0.5 to 2 mm or a little piece of
sacrificial column.
Injection => Precolumn => Column => Detector

Valve
Prevents the contamination of the expensive analytical columns

with fine particles that can eventually clog the mobile phase
flow.
Analytical Columns
• Common configuration to the right.
• Generally stainless steel and teflon components.
• The stationary phase packings are microporous silica 2-
10 mm in diameter.
• Unmodified silica is very polar.
Columns
There are various columns that are secondary to the separating
column or stationary phase. They are: Guard, Derivatizing,
Capillary, Fast, and Preparatory Columns.
Guard Columns are placed anterior to the separating column. This
serves as a protective factor that prolongs the life and usefulness of
the separation column. They are dependable columns designed to
filter or remove: 1) particles that clog the separation column; 2)
compounds and ions that could ultimately cause "baseline drift",
decreased resolution, decreased sensitivity, and create false peaks; 3)
compounds that may cause precipitation upon contact with the
stationary or mobile phase; and 4) compounds that might co-elute
and cause extraneous peaks and interfere with detection and/or
quantification. These columns must be changed on a regular basis in
order to optimize their protective function. Size of the packing
varies with the type of protection needed.
Other types of columns used in HPLC
• Guard column:
• 1- Protect the
analytical column
• 2- Organization of
separation in HPLC
Derivatizing Columns- Pre- or post-primary column derivatization can be
an important aspect of the sample analysis. Reducing or altering the parent
compound to a chemically related daughter molecule or fragment elicits
potentially tangible data which may complement other results or prior analysis.
In few cases, the derivatization step can serve to cause data to become
questionable, which is one reason why HPLC was advantageous over gas
chromatography. Because GC requires volatile, thermally stabile, or nonpolar
analytes, derivatization was usually required for those samples which did not
contain these properties. Acetylation, silylation, or concentrated acid hydrolysis
are a few derivatization techniques.
Capillary Columns- Advances in HPLC led to smaller analytical columns.

Also known as microcolumns, capillary columns have a diameter much less than
a millimeter and there are three types: open-tubular, partially packed, and tightly
packed. They allow the user to work with nanoliter sample volumes, decreased
flow rate, and decreased solvent volume usage which may lead to cost
effectiveness. However, most conditions and instrumentation must be
miniaturized, flow rate can be difficult to reproduce, gradient elution is not as
efficient, and care must be taken when loading minute sample volumes.
D- Column
Column in HPLC is either
• 1- Analytical, 1-6 mm i. d.
• 2- Preparative up to 3 cm
i. d.
• Made from: Stainless
• Shape: Straight
• Length: Variable
Different shapes for columns

used in HPLC
Microbore and small-bore columns are also used for analytical and small
volumes assays. A typical diameter for a small-bore column is 1-2 mm. Like
capillary columns, instruments must usually be modified to accommodate these
smaller capacity columns (i.e., decreased flow rate). However, besides the
advantage of smaller sample and mobile phase volume, there is a noted increase
in mass sensitivity without significant loss in resolution. ---Capillary
Electrophoresis
Fast Columns- One of the primary reasons for using these columns is to obtain
improved sample throughput (amount of compound per unit time). For many
columns, increasing the flow or migration rate through the stationary phase will
adversely affect the resolution and separation. Therefore, fast columns are
designed to decrease time of the chromatographic analysis without forsaking
significant deviations in results. These columns have the same internal diameter
but much shorter length than most other columns, and they are packed with
smaller particles that are typically 3 µm in diameter. Advantages include
increased sensitivity, decreased analysis time, decreased mobile phase usage,
and increased reproducibility.
Preparatory Columns- These columns are utilized when the objective is to prepare
bulk (milligrams) of sample for laboratory preparatory applications. A preparatory
column usually has a large column diameter which is designed to facilitate large
volume injections into the HPLC system.
Accessories important to mention are the back-pressure regulator and the fraction
collector. The back-pressure regulator is placed immediately posterior to the HPLC
detector. It is designed to apply constant pressure to the detector outlet which
prevents the formation of air bubbles within the system. This, in turn, improves
chromatographic baseline stability. It is usually devised to operate regardless of
flow rate, mobile phase, or viscosity.The fraction collector is an automated device
that collects uniform increments of the HPLC output. Vials are placed in the
carousel and the user programs the time interval in which the machine is to collect
each fraction. Each vial contains mobile phase and sample fractions at the
corresponding time of elution. Packings for columns are diverse since there are
many modes of HPLC. They are available in different sizes, diameters, pore sizes,
or they can have special materials attached (such as an antigen or antibody for
immunoaffinity chromatography). Packings available range from those needed for
specific applications (affinity, immunoaffinity, chiral, biological, etc.) to those for
all-purpose applications. The packings are attached to the internal column hull by
resins or supports, which include oxides, polymers, carbon, hydroxyapatite beads,
agarose, or silica, the most common type(Brown, 1990).
Column Heaters in HPLC
• Heating the column in HPLC will improves mass transport,
decreases the Cu term in the van Deemter equation.
• Consider the following example:
• Notice that the tr for each solute changes with temperature, this is
because of the solubility changes we should expect with T.
HPLC Column Ovens
• Block heater with solvent preheater
– Column is housed between 2 metal plates
– Mobile phase is plumbed into the block for
preheating
• Forced air
– Column is in a large chamber with air circulation
– Better temperature equilibration
– Room for column switching valves
Column Oven
Retention times decrease, and higher flow rates are possible

Constant temperature for solvent and column is required to perform reproducible
results.
Column Care
• Follow MFR’s recommendations for solvent
compatibility, flow rate, and pressure limits
• Filter samples when possible
– Particulates will build up on the inlet frit over time
• Use care when reversing column flow
– Connect the outlet to waste, NOT inline with the
detector to prevent further contamination
• Store columns in recommended solvents
Stationary Phase
The stationary phase in HPLC refers to the solid support contained
within the column over which the mobile phase continuously flows. The
sample solution is injected into the mobile phase of the assay through the
injector port. As the sample solution flows with the mobile phase through
the stationary phase, the components of that solution will migrate
according to the non-covalent interactions of the compounds with the
stationary phase. The chemical interactions of the stationary phase and the
sample with the mobile phase, determines the degree of migration and
separation of the components contained in the sample. For example, those
samples which have stronger interactions with the stationary phase than
with the mobile phase will elute from the column less quickly, and thus
have a longer retention time, while the reverse is also true. Columns
containing various types of stationary phases are commercially available.
Some of the more common stationary phases include: Liquid-Liquid,
Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase,
Ion Exchange, and Affinity.
Liquid-Solid operates on the basis of polarity. Compounds that possess
functional groups cabable of strong hydrogen bonding will adhere more tightly to
the stationary phase than less polar compoounds. Thus, less polar compounds will
elute from the column faster than compounds that are highly polar.
Liquid-Liquid operates on the same basis as liquid-solid. However, this

technique is better suited for samples of medium polarity that are soluble in
weakly polar to polar organic solvents. The separation of non-electrolytes is
achieved by matching the polarities of the sample and the stationary phase and
using a mobile phase which possesses a markedly different polarity.
Size-Exclusion operates on the basis of the molecular size of compounds

being analyzed. The stationary phase consists of porous beads. The larger
compounds will be excluded from the interior of the bead and thus will elute first.
The smaller compounds will be allowed to enter the beads and will elute
according to their ability to exit from the same sized pores they were internalized
through. The column can be either silica or non-silica based. However, there are
some size-exclusion that are weakly anionic and slightly hydrophobic which give
rise to non-ideal size-exclusion behavior.
Normal Phase operates on the basis of hydrophilicity and lipophilicity by
using a polar stationary phase and a less polar mobile phase. Thus hydrophobic
compounds elute more quickly than do hydrophilic compounds.
Reverse Phase operates on the basis of hydrophilicity and lipophilicity. The

stationary phase consists of silica based packings with n-alkyl chains covalently
bound. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand
in the matrix. The more hydrophobic the matrix on each ligand, the greater is the
tendancy of the column to retain hydrophobic moieties. Thus hydrophilic
compounds elute more quickly than do hydrophobic compounds.
Normal-phase Reversed-phase
chromatography chromatography
Polar stationary phase nonpolar stationary phase
More polar solvent has Less polar solvent has

higher eulent strength higher eulent strength
Ion-Exchange operates on the basis of selective exchange of ions in the
sample with counterions in the stationary phase. IE is performed with columns
containing charge-bearing functional groups attached to a polymer matrix.
The functional ions are permanently bonded to the column and each has a
counterion attached. The sample is retained by replacing the counterions of
the stationary phase with its own ions. The sample is eluted from the column
by changing the properties of the mobile phase do that the mobile phase will
now displace the sample ions from the stationary phase, (ie. changing the pH).
Affinity operates by using immobilized biochemicals that have a specific

affinity to the compound of interest. Separation occurs as the mobile phase
and sample pass over the stationary phase. The sample compound or
compounds of interest are retained as the rest of the impurities and mobile
phase pass through. The compounds are then eluted by changing the mobile
phase conditions.
Stationary Phases (Adsorbents)
HPLC separations are based on the surface interactions, and depends on the types of
the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small
rigid porous particles with high surface area.
Main adsorbent parameters are:
• Particle size: 3 to 10 mm
• Particle size distribution: as narrow as possible, usually within 10% of the mean;
• Pore size: 70 to 300 mm

• Surface area: 50 to 250 m2/g
• Bonding phase density (number of adsorption sites per surface unit): 1 to 5 per 1
nm2
The last parameter in the list represents an adsorbent surface chemistry. Depending
on the type of the ligand attached to the surface, the adsorbent could be normal
phase (-OH, -NH2), or reversed-phase (C8, C18, Phenyl), and even anion (NH4+), or
cation (-COO-) exchangers.
Plate height as a function of flow rate for stationary-phase particle sizes of 10, 5, and 3mm.
The Stationary Phase
Most common support –highly pure, spherical, microporous particles of permeable
silica, several hundred m2/g
Scanning electron micrographs of silica chromatography

particles.
(a) Aggregate of spherical particles (50% porosity, 150
m2/g surface area)
(b) Spongelike structure (70% porosity, 300m2/g surface
area)
Schematic structure of sillica gel.
The configuration of packed materials. (a)Large porous (b) pellicular
(c) Porous microparticle
The configuration of inside HPLC column
Flow I.D 100 ~ 700 mm
Stationary phase
Wall of column
Solid support
(a) Wall coated open tube (b) Support coated open tube (c) Porous layer open tube
Types of open capillary column.

Particle Size
Distribution of several column batches
• Columns have a
distribution of particle
sizes
• Reported “particle
diameter” is an
average
• Broader distribution --
-> broader peaks
Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82
HPLC – Particles Size
HPLC – Porous and Solid Packing
HPLC – Effect of Carbon Chain Length on Separation
HPLC – Column Diameter and Separation
Size Exclusion HPLC
HPLC – Derivatization for Detection
Typical Column Sizes
• Particle size: 5 µm, 3 µm, and smaller
– Monodispersed means particles are the same size
– Very important for stable pressure and flow
– Smaller particles produce higher system pressure
• Pore size: 100-120 A is typical
• Surface area: 300-350 m2/g
• Carbon load: 9-12% for C8, 16-20% for C18
– Higher carbon load = better resolution but longer
run times
– Lower carbon load = shorter run times, but may
change selectivity vs. higher carbon load
Bonded stationary phase
Silica surface dimethyl silane R= C8, C18, ……
The octadecyl (C18) stationary phase is by far the most common in

HPLC. It is frequently designated ODS, for octadecylsilane
Common polar phases Common nonpolar phases

R = (CH2)3NH2 amino R = (CH2)17CH3 octadecyl
R = (CH2)3C≡N cyano R = (CH2)7CH3 octyl
R = (CH2)2OCH2(OH)CH2OH diol R = (CH2)3C6H5 phenyl

Bulky isobutyl groups protect siloxane bonds
Molecular model of octadecyl from hydrolysis at low pH. Siloxane (Si-O-
SiR) bond hydrolyze below pH2 PH 2-8
siloxane
Mechanism of separation in different forms of
HPLC
Hydrophobic interaction chromatography
Two stationary phases or hydrophobic interaction chromatography.

Separation mode
Column and mobile phase solvent

3. Separation mode
Sample and Analytical method
What is the sample ?

Concentration of the interested component
Contaminant
In which materials ?
In what concentration ? Characteristics of the sample
- Structure
Which sample ? - Molecular weight
With which technique ? - pKa
- Solubility
Analytical technique
- Column
- Mobile phase
- Detector
- Sample preparation
3. Separation mode
Sample information
Merck Index
Great Chemical Dictionary
Great BioChemical Dictionary
Reports based on other measurement
techniques
3. Separation mode
Method information
Society magazines
Journal of Chromatography.
Analytical Chemist
Manufacturer
JASCO Application data
3. Separation mode
HPLC separation mode
HPLC separation mode
Normal phase chromatography (NP)

Reversed phase chromatography (RP)
Size exclusion chromatography (SEC)
Ion exchange chromatography (IEX)
Affinity chromatography
3. Separation mode
Separation modes and features
Mode Stationary phase Mobile phase Interaction Feature
Normal phase Silica gel Organic solvent Adsorption Fat-soluble

chromatography (n-Hexane/IPE)
Reversed phase Silica-ODS MeOH/Water Hydrophobic Most widely used

chromatography (Silica-C18)
Size exclusion Chromatography

Non-aqueous (GPC) Porous Polymer Organic solvent (THF) Gel permeation Molecular weight distribution
Aqueous (GFC) Aqueous porous Polymer Buffer solution Gel permeation Protein Separation
Ion exchange Ion exchange gel Buffer solution Ion exchange Separation of
Chromatography ionic substances
Affinity Packing with ligand Buffer solution Affinity Purification of

Chromatography enzymes and proteins
GPC : Gel Permeation Chromatography

GFC : Gel Filtration Chromatography
3. Separation mode
Solvent used in HPLC and range of Application

Solvent Polarity E0 R.I. b.p. Viscosity UV cut off UV transmittance
200 250 300 350
Isoctane 0.1 0.01 1.389 99 0.47 197
LCn-Hexane 0.1 0.01 1.372 69 0.30 190
Cyclohexane -0.2 0.04 1.423 81 0.90 200
Triethylamine 1.9 0.54 1.398 89 0.36
i-Proryl ether 2.4 0.28 1.365 68 0.38 220*
Toluene 2.4 0.29 1.494 110 0.55 285
Ethyl ether 2.8 0.38 1.350 35 0.24 218
Benzene 2.7 0.32 1.498 80 0.60 280
Methylene chloride 3.1 0.42 1.421 40 0.41 233
n-Butanol 3.9 0.7 1.397 118 2.6 210
n-Propanol 4.0 0.82 1.385 97 1.9 240
Tetrahydrofuran 4.0 0.57 1.405 66 0.46 212*
Ethyl acetate 4.4 0.58 1.370 77 0.43 256
i-Propanol 3.9 0.82 1.384 82 1.9 205
Chloroform 4.1 0.40 1.443 61 0.53 245
Methylethyl ketone 4.7 0.51 1.376 80 0.38 329
Dioxane 4.8 0.56 1.420 101 1.2 215
Acetone 5.1 0.56 1.356 56 0.30 330
Ethanol 4.3 0.88 1.359 78 1.08 210
Acetic acid 6.0 1.370 118 1.1
Acetonitrile 5.8 0.65 1.341 82 0.34 190
Dimethylformamide 6.4 1.428 153 0.80 268
Dinethylsulfoxide 7.2 0.75 1.477 189 2.00
268Methanol 5.1 0.95 1.326 65
0.54 205
Water 10.2 1.333 100 0.89
3. Separation mode
Polar compounds Non-polar compound

Polar compound
H
H H
H C H
O
Bonding electrons are not shared evenly. H
The end of the bond with electrons becomes partially negative.
The end of the bondwithout electrons becomes partially positive.
Polar compounds are soluble in polar solvents.

Non-polar compounds are soluble in non-polar solvents.
normal and reversed-phase chromatography.
• In normal phase chromatography, the stationary bed is strongly polar in
nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or
tetrahydrofuran). Polar samples are thus retained on the polar surface of the
column packing longer than less polar materials.
• Reversed-phase chromatography is the inverse of this. The stationary bed is
nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such
as mixtures of water and methanol or acetonitrile. Here the more nonpolar the
material is, the longer it will be retained.
Above mentioned types cover almost 90% of all chromatographic applications.
Eluent polarity plays the highest role in all types of HPLC.
There are two elution types: isocratic and gradient. In the first type constant
eluent composition is pumped through the column during the whole analysis. In
the second type, eluent composition (and strength) is steadily changed during
the run.
Separation Principles in HPLC
General Rule of Thumb:
Polarity of analytes ≈ polarity of stationary phase ≠
polarity of mobile phase
To achieve good separation, the analytes should

interact with the stationary phase, but not too
strongly or the retention time will become very
long
Reversed order
of elution
Increasing Mobil
phase Polarity,
Decreases
Elution Time
Normal phase
Reversed Phase
How to Increase HPLC Resolution
1. Increase column length

2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution
3. Separation mode
Normal Phase Chromatography
Interaction : Adsorption
Packing materials : Polar ex. Silica gel

Silica-NH2
Silica-CN
Silica-OH
Mobile phase : Non-polar ex. n-Hex/CH2CL2

iso-Oct/IPA
iso-Oct/AcOEt
Sample : Fat-soluble
Different polarity
3. Separation mode

Packing material
The most popular packing material is silica gel.
It is believed that silanol radicals ( -Si-OH ) on the surface of silica gel
act as the active site and the sample is separated.
Si
Si Si
the surface of silica gel

3. Separation mode
Interaction
OH H2N
OH O2N
OH H2N NO2
OH H2N
OH O2N
OH
OH
3. Separation mode

Mobile phase solvents
n-Hexane （n-Hex）
Low
iso-Octane （iso-Oct）
Chloroform （CHCl3）
Dichloromethane （CH2Cl2）
Ethylacetate （AcOEt）
Isopropylalchol （IPA）
Tetrahydrofran （THF） Polarity
Dioxane
Acetonitrile （CH3CN）
Ethanol （EtOH）
Methanol （MeOH）
Amines
Acids High
3. Separation mode

Retention behavior
Low
A B C
n-Hex/AcOEt(60/40)
A B C D
Polarity of
Mobile phase n-Hex/AcOEt(50/50)
A BC
D
Polarity of sample components
A < B < C < D

High n-Hex/AcOEt(30/70)
Normal Phase LC
– Polar stationary phase: Silica
– Nonpolar mobile phase: Hexane, Ethyl acetate
– The LEAST polar compound comes out first
Normal Phase HPLC Columns
• Cyano: Rugged, moderate polarity,
general use
• -OH (Diol): More polar and retentive
• Amino: Highly polar, less stable
• Silica: Very rugged, low cost,adsorbent
(Unbonded)
The cyano column with a low polarity mobile phase (hydrocarbon with a
amall amount of another solvent) will act as a normal phase column.
Normal Phase Column Chromatography
The column packing in the column is very polar!
Polar compounds are going to be attracted to the polar

column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column first,

while the polar compounds are going to come off column last.
Usually, one starts will a less polar solvent to remove

the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
3. Separation mode
Reversed Phase Chromatography
Interaction : Hydrophobic
Packing materials : Non-polar ex. Silica-C18

Silica-C8
Polymer
Mobile phase : Polar ex. MeOH/H2O

CH3CN/H2O
MeOH/Buffer sol.
Sample : Having different length of carbon chain

3. Separation mode

CH3
O－Si－CH2(CH2)16CH3
Silica-C18 Packing materials CH3

Si
Commonly used packing materials are hydrocarbons CH3
having 18 carbon atoms (called the Octadecyl radical)
which are chemically bonded to silica gel (Silica-
ODS).Since the surface of the Silica-ODS is covered
with hydrocarbon, the polarity of the packing material
CH3
itself is very low.
CH3
O－Si－CH3
Si CH3
CH3
CH3
3. Separation mode

Hydrophobic Interaction
CH3 CH2COOCH3
CH3 CH2COOCH3
Silica-C18 (ODS)
3. Separation mode
Mobile phase solvents

Main solvent : MeOH - H2O
CH3CN - H2O
Sub solvent : EtOH

IPA
THF
DMF
Additive : Acid
Salt
Ion-pairing agent
3.Separation mode
Retention behavior in reversed phase HPLC

CH3CN/H2O
(70/30) (60/40) (50/50)
A
B A
C B A
C B
C
Carbon chain length of sample
A < B < C
A : p-Hydroxy ethyl benzoate 0 5 0 5 0 5 10 (min)

B : p-Hydroxy propyl benzoate
C : p-Hydroxy butyl benzoate Low Polarity of Mobile phase High
Column : Finepak SIL C18
3.Separation mode
Length of packing materials carbon chains
and retention time A
Finepak SIL C18 B
C
A
B
Finepak SIL C8
C
A
Mobile phase：CH3CN/H2O(40/60) B
Finepak SIL C1 C
A : p-Hydroxy ethyl benzoate
B : p-Hydroxy propyl benzoate
C : p-Hydroxy butyl benzoate
0 5 10 15 20 25 30 (min)
3.Separation mode
Reversed Phase and Normal Phase Chromatography
Comparison of Reversed Phase and Normal Phase
Normal phase Reversed phase
Stationary phase High polarity Low polarity
Mobile phase Low polarity High polarity
Interaction Adsorption Hydrophobic
Elution order Low to High Short to Long

(Polarity) (Length of Carbon chain)
3.Separation mode
Reversed Phase and Normal Phase Chromatography
Comparison of
Reversed Phase and Normal Phase Reversed
Reversed Phase
Phase
Chromatography
Chromatography
Finepak
FinepakSIL
SILC18
C18
MeOH
MeOH
VA
VD
VA
Normal
Normal Phase
Phase
Chromatography
Chromatography
Finepak
FinepakSIL
SIL
VD
VE
n-Hexane/IPA(96/4)
n-Hexane/IPA(96/4)
0 10 (min) 0 10 20 (min)
Reversed-Phase LC
– Nonpolar stationary phase: C8, C18
– Polar mobile phase: Water, ACN, Methanol
– The MOST polar compound comes out first
Reversed Phase HPLC Columns
• C-18, C-8: Rugged, general purpose, highly
retentive
• C-3, C-4: Less retentive, used mostly for peptides
& proteins
• Phenyl: Greater selectivity than alkyl-bonded
• Cyano: Moderate retention, normal & rev.
phase
• Amino: Weak retention, good for carbohydrates
The cyano column with a high polarity mobile phase (Water/MeOH) will act
as a reversed phase column.
Reverse Phase
Column Chromatography
• The stationary phase (column packing) is now NON-
POLAR
• Non-polar compounds will move more slowly because
they are attracted to the column packing.
• The more polar component moves more quickly down
the column.
• Polar solvents, such as water and methanol are used in
reverse phase chromatography
• Used mainly in columns, such as HPLC
Common RP Packings
RP Column Properties
• Hydrophobic Surface
• Particle Size and Shape
• Particle Size Distribution
• Porosity, Pore Size and Surface Area
RP Mechanism (Simple)
3.Separation mode
Ion-exchange Chromatography
Interaction : Ion-exchange
Stationary phase: Anion exchange gel

Cation exchange gel
Mobile phase : Buffer solution
Sample : Ionic substances (Cations or Anions)

3.Separation mode
Ion-exchange Gel
SO3- Na +
NR3+ Cl -
NR3+ Cl -
SO3- Na +
+ Cl -
SO3- Na + NR3
SO3- Na + NR3+ Cl -
Cation exchange gel Anion exchange gel

3.Separation mode
Mobile phase solvents used for Ion-exchange
Buffer solution
Salt concentration
pH (Hydrogen ion concentration) SO3 - Na +
Type of salt
Additive (Organic solvent) S+
SO3 -
Na +
Na +
SO3 -
S +
3.Separation mode
Application data of Ion-exchange chromatography
Separation of polyphosphoric acid
Column : Finepak GEL SA-121
1 5.89
1. 2E+05
uV (6.0mmI.D. POLY_003.CH2
x 100mmL)
2 9.49
Mobile phase : A= 0.1M KCl + 1% EDTA-4Na
(pH 10.0 adjusted HCl)
B= 1.0M KCl + 1% EDTA-4Na
1. 0E+05
(pH 10.0 adjusted HCl) gradient
Reactor : 1.8MH2 SO4(1L),
(NH4o7)6MO24-4H2O (5g),
4 17.79
5 21.65
Sand of zinc metal(0.6g)
3 13.58
6 24.65
Detection : 830nm
7 26.95
8. 0E+04
8 28.83
9 30.40
6. 0E+04
11 32.86
10 31.71
12 33.84
4. 0E+04
13 34.72
14 35.50
15 36.21
16 36.85
17 37.43
2. 0E+04
18 37.95
19 38.38
20 38.72
0. 0E+00
10.0 20.0 30.00 40.00 [m in]

0 0
3.Separation mode
Ion Chromatography
Summary of Ion Chromatography

Purpose : Separation of inorganic ions, organic acids
Stationary phase: Anion exchange gel

Cation exchange gel
Detection : Conductivity detector

3.Separation mode
Ion Chromatography
Suppressor and Non-suppressor
P pump
P pump
Mobile phase Mobile phase

injector injector
column column
suppressor
D
Conductivity
detector
D Conductivity
detector
3.Separation mode
Ion Chromatography
Cation measurement data
Sample : Tap water

Column : Shodex IC YK-421
Na+ 5.276ppm
Mobile phase : 5mM tartaric acid+2mM Gibicolin acid
Detector : Conductivity detector (CD-5)
Ca2+ 12.386ppm
Mg2+ 1.829ppm
K+ 0.785ppm
0 5 10 15 20(min)
3.Separation mode
Ion Chromatography
Anion measurement data
Sample : Tap water

Column : Shodex IC I-524A
Cl- 6.029ppm
Mobile phase : 2mM phthalic acid+1.84mM tris
+300mM boric acid(pH4.0)
Detector : Conductivity detector (CD-5)
SO42- 10.426ppm
NO3- 6.694ppm
F- 0.111ppm
0 5 10 15 20 25(min)
3.Separation mode
Size Exclusion Chromatography (SEC)
GPC and GFC
Non-aqueous SEC : GPC (Gel Permeation Chromatography)

Interaction : Gel permeation
Packing : Cross-Linked porous Polystyrene
Mobile phase : Organic solvent (THF, CHCl3, DMF)
Sample : Molecular weight distribution of polymer
Synthetic Oligomer separation
Aqueous SEC : GFC (Gel Filtration Chromatography)

Interaction : Gel permeation
Packing : Hydrophilic silica gel / Hydrophilic porous polymer
Sample : Separation of Water-soluble polymers
(proteins, nucleic acid, sugar)
oligomers
3.Separation mode
SEC Separation mechanism
B
Small pore
Mobile phase A
D
A D
C Packing material
C
D
C
B
D
A+B C
3.Separation mode
Gel permeation chromatography and calibration curve
uV RI
1. 0E+05
Column : Shodex GPC KF-806Lx 2 Column

Mobile phase : THF
8. 0E+04
PS-18.1K
PS-110K
PS-Oligomer
PS-2.98K
6. 0E+04
PS-900K
PS-8420K
4. 0E+04
2. 0E+04
0. 0E+00
5. 00 10. 00 15. 00 20. 00 25. 00 30. 00 35. 00[ mi n]

3.Separation mode
Peak analysis of polymer
to calculate molecular weight distribution
no Vi Hi
1 10.0 74
2 10.5 156
3 11.0 318
H2 H3
H1
10.0 15.0 20.0 25.0 (min)

V1
Retention time
V 2 V3
3.Separation mode
Molecular weight calculation
N Vi Hi Mi Hi/Mi HiMi HiMi2

1 11.12 74 2094050 - - -
2 11.37 387 1734413 - - -
3 11.62 1539 1432619 - - -
- - - - - - -
- - - - - - -
- - - - - - -
hi mi Hi/Mi HiMi HiMi2
Mn = Hi/ Hi/Mi = 15.5×104

Mw = HiMi/ Hi = 28.6×104
Mz = HiMi2/ HiMi = 46.5×104
D = Mw/Mn = 1.84
3.Separation mode
Column selection
Molecular weight of the sample : Exclusion limit molecular weight
Ability to dissolve the sample : Applicable to packing materials
Molecular weight distribution : Range of calibration curve

3.Separation mode
Column suited to the sample in terms of molecular weight
Shodex A-801×2 Shodex A-803×2
2 1
EPIKOTE 2 1 3 n=0
1001 3-8 n=0 4
5
6
7
8
EPIKOTE 1 1
828
2 2
30 40 min
15 20 25 min
Eluent : THF Flow rate : 1.0ml/min

3.Separation mode
Solvent and column
Solvent Column
THF Finepak GEL 101F

Shodex KF series
CHCl3 Finepak GEL 101C

Shodex K series
DMF Shodex KD series
H2O, Buffer solution Shodex SB series

Shodex KS series
3.Separation mode
Calibration curves for columns
Eluent : THF
3.Separation mode
Columns for exclusive use
Columns for Exclusive use

Amino acids : Aapak (Cation exchange)
Organic acid : Shodex Ionpak KC-811

(ion exclusion and partition & adsorption)
Sugar : Shodex Ionpak KS series (aqueous SEC)

Shodex Sugar series (ligand exchange)
Finepak SIL NH2-5 (Normal phase)
Finepak GEL SA-121 (Strong anion exchange)
N-methyl carbamate : Carbamatepak (Reversed phase)

3.Separation mode
Amino Acid Analysis

Column : AApak Na II-H
Ala
Mobile phase : Sodium citrate buffer
Stepwise gradient
Detection : OPA post label
Gly
Ex 345nm Em 445nm
Val
Sample : Sake
NH3
Glu
ThrSer
Leu
Pro
Arg
His
Asp
Ile
Lys
Phe
Tyr
Met
3.Separation mode
Organic Acid Analysis

Column : Shodex Ionpak KC811x2
Mobile phase :
lactic
Detection : BTB post label
UV 445nm
succinic
citric Sample : Sake
pyrvic
pyroglutamic
acetic
malic
3.Separation mode
Ion suppression method & Ion-pair chromatography

Separation method to analyze ionic compounds by reversed-phase
chromatography
Ion suppression method : Acidic ion components
Ion pair chromatography : Basic ion components / Acidic ion components

3.Separation mode
Diagram of Ion suppression method
Add phosphoric acid
H+
H+
H+ A- HA
H+
HA
A- H+
Silica-C18 Silica-C18
H+ H+
H+
A- HA
H+
H+ H+
A- + H+ HA
A- : Sample
H+ : Hydrogen ion
HA : Sample
3.Separation mode
Chromatogram when Ion suppression method is used
p-Hydroxy ethyl benzoate

Benzoic acid
Finepak SIL C1 Finepak SIL C1
CH3CN/H2O CH3CN/0.2% H3PO4
(40/60) (40/60)
propyl
butyl
0 5 10 (min) 0 5 10 (min)
3.Separation mode
Diagram of Ion-pair chromatography
SO3-
+NR SO3-
4 Silica-C18
SO3-
+NR Add Ion-pair

4
Silica-C18 reagent
SO3-
+NR
4
Silica-C18 SO3- + NR4
3.Separation mode
Chromatogram when Ion-pair chromatography is used

A
A
B B
Without Ion-pair reagent With Ion-pair reagent
Typical ion reagents

Acidic ions : Tetra alkyl ammonium halide
Basic ions : l-Alkyl sulfonate
3.Separation mode
Ion-pair chromatography
Effects of basic additives

- Stable pH
- Longer retention time
- Ion pair reagent effect
3.Separation mode
Acid and basic sample for Reversed phase LC
Method Sample Reversed phase
Ion suppression Weak acidic sample phosphoric acid

acetic acid
perchloric acid
trifluoroacetic acid
Ion pair Acidic sample Tetra alkyl ammonium halide

Basic sample l-Alkykl surfonate
(acetic acid)
(trifluoroacetic acid)
Addition of salt phosphate

citrate
Band Broadening in LC
• Back to the van Deemter Equation,
H = A + B/u + Cu
• Which of the three components is the largest contribution

to H? Consider the following:
• B/u effects – Diffusion is usually 100x less in liquids than in

the gas phase.
• Cu effects – By process of elimination we will assume that

mass transport effects are the largest contribution to H in
LC.
Cu – Mass Transfer (MT) Effects.
• This is the effect of the kinetics of mass transfer to/from the mobile
phase to/from the stationary phase.
Review of MT effects
f ( k ' ) d p2
Cm 
MT in the m.p. Dm
Where dp is the diameter of the packing particle in LC,
Stationary
Mobile phase
Phase Flow
Packing particle
(silica)
Smaller dp increases the surface area/volume ratio and thus

increases M.T. in the m.p.
Smaller dp increases the surface area/volume ratio
and thus increases M.T. in the m.p.
• Volume = 4/3  r3 Surface Area = 4  r2
• Surface area/volume = 1/3r
• The effect is dramatic in figures 25-2 & 25-3
• The cost of small packing particles is that the pressure required to force
liquid through the column follows as:
P  1/dp3
• The typical particle sizes in HPLC is 3-10 mm. In order to achieve flow
rates of 0.5 to 5 mL/min, for a 10-30 cm column, pressures of 70 to 400
atm (1000 to 6000 psi) are required.
Figure 25-3
Figure 25-2
Separations
Injector
Separation in based upon differential
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Pumps Mobile Phase - carries the sample

through the stationary phase as it
moves through the column.
Column
Detector
Waste
Solvents
High Performance Liquid Chromatograph

Separations
Injector Chromatogram
Mixer mAU
Pumps
Start Injection time
Column
Detector
Solvents
High Performance Liquid Chromatograph

Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
Separations
Mixer mAU
Pumps
Column
Detector
Solvents
The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR
tR
mAU Area or height is proportional
to the quantity of analyte.
to
Injection time
261
6. Data processing
Data processing in HPLC
1. Qualitative analysis
2. Quantitative analysis
3. Molecular weight distribution
6. Data processing
Qualitative analysis
1. Retention time
2. Retention volume of the standard sample
3. Sample components are collected after separation,
and subjected to spectrometric analysis
such as IR, NMR and MS.
6. Data processing
Identification from retention time
tR Standard sample
Unknown sample
A B
6. Data processing
Standard addition method
Standard addition
Target peak
6. Data processing
Standard addition method
Retention time of standard sample
is different from unknown sample
Standard sample
Unknown sample
Unknown sample and

Standard sample
6. Data processing
Identification using a different instruments
after preparative analysis
Identification from retention time
Limitation:
On flow UV spectrum
On flow emission spectrum
Multi-channel detector
Preparative analysis
Spectrum measurement using
a different instrument
6. Data processing
Quantitative analysis
How much component A ?
A
Standard sample (1mg/ml)
The amount of a
component can be
calculated from the peak Injection
height and peak area of the of 10μg
chromatogram.
Unknown sample
A
Injection
of 10μg
6. Data processing
Calibration method
External standard sample
Internal standard sample
6. Data processing
External standard sample

Thiamiral in serum
Thiamiral
Peak area
Thiamiral
Concentration(μg/ml)
Finepak SIL C18T-5、 CH3CN/10mM KH2PO4 aq. (50:50)

UV 288nm
6. Data processing
Internal standard sample

Anticonvulsants in serum
Standard sample Calibration curve Unknown sample

1：PB
2：DPH
3：CBZ
IS：Phenacetin
Peak area
s
concentration(μg/ml)
Concentration ratio
Finepak SIL C18T、 CH3CN/5mM KH2PO4 aq.

6. Data processing
Guide for selecting the internal standard sample
No overlapping peaks
No Components included in unknown sample
Chemical and physical stability
High purity
6. Data processing
External standard and Internal standard samples

External standard Internal standard
Error injection volume volume to be added
Correction of impossible possible
Pre-treatment loss
6. Data processing
Caution when using an Integrator

One point calibration Integrator
True curve
error large small large

6. Data processing
Caution when using an Integrator

Two point calibration
Integrator
True curve
error large small large

6. Data processing
Baseline
6. Data processing
Considerations when performing quantitative analysis

Standard sample
Integrator
Micro syringe
Sample preparation
Concentration change of standard sample
Contamination
The JASCO advanced technology team has again met the challenge and designed a new
line of HPLC instruments, The LC-1500series more than satisfies in response to the
growing demand for greatly expanded HPLC analyses in the fields of not only
biochemistry, pharmaceutical and medical science, but also in the areas of among other
organic and inorganic compounds, foods, agricultural sciences, polymeric and natural
substances and pollution. The LC-1500 series comprises pumps, detectors,
autosamplers, its own column oven and other units each having built-in intelligence and
incorporating many features with much higher levels of operability and reliability in
addition to multiple functions, higher performance and higher accuracy than before,
making them the most advanced instruments available.
7. Sample preparation
Sample preparation
Cause Problem Countermeasures

Sample is not liquid. not possible to inject extraction / dissolving
Concentration is too high. over load for column / out of detection range dilution
Concentration is too low. cannot detect concentration / derivative
Contains foreign particles clogged up centrifugation / filtration
Includes components which damage column solvent extraction /derivative
Includes interference for separation quantitation error solvent extraction /derivative
Solvent unsuitable deterioration of column pH adjustment
Sample preparation Method

Filtration 0.45um, 0.2um membrance filter
Extraction Solvent extraction
Solid phase extraction
Concentration Evaporation
Solid phase exraction (Bond Elut)
Fused drying
Deprotaination Organic acid
Homonization
Solid phase extraction 7. Sample preparation
1. Activation 2. Load sample 3. Wash 4. Elute target compound
Activate Wash with H2O MeOH or Eluting

Wash with Sample orH2O/MeOH solvent
With H2O Contaminant
MeOH
Target
Contaminant compound
Vacuum
Removing contaminants
which have strong retention 7. Sample preparation
1. Activate 2. Load sample 3. Elute a target compound
Wash with Activate with

proper solvent Target sample
MeOH Using vacuum or pressure
Compound which
has strong retention
Compound which
has strong retention
Target sample
Vacuum
Concentration 7. Sample preparation
1. Activate 2. Load and concentrate 3. Elute target sample
target sample
Wash with （ target sample）

Activate with
MeOH H2O Elute with MeOH
pump
Small amount of
Target sample
Target compound
is concentrated.
Vacuum
Considerations when preparing sample

Recovery rate
Contamination
The JASCO advanced technology team has again met the challenge and designed a new
line of HPLC instruments, The LC-1500series more than satisfies in response to the
growing demand for greatly expanded HPLC analyses in the fields of not only
biochemistry, pharmaceutical and medical science, but also in the areas of among other
organic and inorganic compounds, foods, agricultural sciences, polymeric and natural
substances and pollution. The LC-1500 series comprises pumps, detectors,
autosamplers, its own column oven and other units each having built-in intelligence and
incorporating many features with much higher levels of operability and reliability in
addition to multiple functions, higher performance and higher accuracy than before,
making them the most advanced instruments available.
8. Procedure for developing analytical conditions

Procedure for developing analytical conditions
Step one : clear analytical purpose, and research the target

compound.
(1) Molecular weight
Molecular structure
Functional group
(2) Solubility, stability
UV, FP absorption
(3) Amount of concentration, contaminant
(4) Application data
reference literature, magazines
Step two : Development analytical conditions

(trial and error)
(1) When attempting to develop analytical conditions,
use an appropriate concentration of standard
solution
(2) Check the detection limit and detection method
(3) Prepare sample
(4) Check contaminat and target compound peak
separation
Procedure for developing analytical conditions
Step three : Establish analytical condition for

routine analysis
(1) Linearity of calibration curve
(2) Reproducibility of analysis
(3) Check for contaminants that retain
strongly in the column
(4) Check for Correlation with other
methods.
Step Four Routine quantitative analysis

(1) Lifetime of column
(2) Running cost
(3) Develop analytical procedure (SOP)
(4) Check HPLC and column performance.
After optimizing the solvent, you might still need to
improve resolution. To increase resolution, you can
- Decrease flow rate
- Increase column length
- Decrease particle size
Temperature as a variable
- Affect the relative retention of different compounds

- Influential for ionic compounds and molecules with
multiple, polar substituents
Gradient Separations
Dwell volume – the volume between the point at which
solvents are mixed and the beginning of the column
Dwell time – the time required for the gradient to reach the
column
dwell volume (mL)
tD =
flow rate (mL.min)
Measurement of dwell time

Gradient Elution Is a Fine Way to Begin Method Development
- The first run on a new mixture should be a gradient
- If ∆ t/tG > 0.25, use gradient elution.
- If ∆ t/tG < 0.25, use isocratic elution.
- The isocratic solvent should have the composition applied to the
column halfway through the period ∆ t
Steps in developing a Gradient Separation

1. Run a wide gradient (e.g., 5 to 100% B) over 40-60min. From this run,
decide whether gradient or isocratic elution in best.
2. If gradient elution is chosen, eliminate portions of the gradient prior to
the first peak and following the last peak. Use the same gradient time
as in step 1.
3. If the separation in step 2 is acceptable try reducing the gfadient time
to reduce the run time
More difficult approaches
- changing the solvent
- using a longer column
- using a small particle size
- changing the separation phase
APLIKASI KCKT
Contoh Aplikasi
Typical applications of bonded-phase chromatography. (a) Soft-drink additives.
(b) Organophosphate insecticides.
Ion chromatogram of a mixture of cations. Ion chromatogram of a mixture of anions.
Gel-filtration chromatogram for glucose(G), Gel-permeation separation of components in an
fructose(F), and sucrose(S) in canned juices. epoxy resin.
Separation of fullerenes
Seapartion of tocopherols by HPLC with electrochemical detection.
Column: TSKgel ODS-80 (4.6mm, 15cm); Mobile phase: 25mM NaClO4 in acetonitrile;
Flow rate: 0.800 ml/min, Temperature: 35oC; Voltage : 800mV; Pressure: 34 kgf/cm2.
APLIKASI KCKT
Content of HPLC test procedure
• Elution procedure: isocratic or gradient
elution
• Preparation of standards and samples
• Operation procedure: sequence of
injections
• System suitability testing (SST) and
criteria
• Calculations
QOS 2.3.R.2 analytical procedures and validation
summaries
Compendial methods
When claim a compendial method, there should be no change in:
• The type of column i.e the stationary phases
• Detector wavelength
• Components in Mobile phase
• System suitability testing and criteria
Adjustments to ratio of components in mobile phase, flow rate,
column temp, dimension of column, particle size (reduction
only), may be necessary to achieve the system suitability
criteria. The allowable variations for each parameter, see
Int.Ph 1.14.4 or USP general chapter <621>.
System suitability testing (SST)
• Precision:
– Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
– Impurities: in general, RSD ≤ 5% at the limit level,
up to 10% or higher at LOQ, n ≥ 6
• Resolution (R): >2
• Tailing factor/peak asymmetry: (≤ 2)
• Number of theoretical plates (N): column efficiency ≥ 2000
• Gradient elution is one way to increase the N

A SST should contain:
• For Assay:
precision + one or more other parameter
• For impurity test:
resolution + precision + one or more other
parameter
Relative Response Factor (RRF)
Quantitation of Impurities
• Against impurity RS’s: when reference
standard available
• Against API itself
Relative response factor should be considered
• Response factor: the response (e.g. peak area)
of drug substance or related substances per
unit weight.
RF= peak area / concentration (mg/ml)
• Relative response factor (RRF):
RRF=RF impurity / RF API, OR,
RRF=slope impurity / slope API
Rifampicine:
y =31.312 x + 4.963
Rifampicine Quinone:
y = 26.198 x + 1.154
RRF= 26.198 / 31.312
=0.84
• To review:
a) RRF calculation, and
b) if RRF is properly used in the final
calculation for % impurity
If RRF within 0.8-1.2, correction may not be
necessay
• Correction factor= 1/RRF, the reciprocal of the
RRF
Review points for HPLC method
• is the analytical procedure described in detail including all the parameters ?
• is SST well defined to ensure the consistency of system performance?
• The preparation of solutions:
– assay: concentration of reference standard should be close to the sample solution
– impurities: concentration of the reference standards should be close to the limit
• The way of quantitation of impurities
In case API is used as the reference, RRF should be used or justification of exclusion
should be provided.
To check the determination of RRF, check the correction of calculation of impurities
• confirm/complete the QOS 2.3.R.2
Validation – compendial methods
Assay – API
No validation generally required. Exception: specificity for major
impurities not in the monograph.
Assay – FPP
Specificity, accuracy and precision (repeatability).
Purity – API and FPP
Full validation for specified impurities that are not included in the
monograph (specificity, linearity, accuracy, repeatability, intermediate
precision, LOD/LOQ)
Validation of the limit for individual unknowns, if tighter than that in the
monograph: LOQ of the API should be below the limit for individual
unknowns
Non-compendial methods
Full validation is required for purity, assay and dissolution methods

(HPLC, UV) :
Specificity
Linearity
Accuracy
Repeatability
Intermediate precision
LOD/LOQ (not required for assay, dissolution)
Robustness (recommended)
Specificity
• Blank solution to show no interference
• Placebo to demonstrate the lack of interference from excipients
• Spiked samples to show that all known related substances are resolved
from each other
• Stressed sample of about 10 to 20% degradation is used to demonstrate
the resolution among degradation products
– Check peak purity of drug substance by photodiode array detector (PDA): eg purity
angle is lower than the purity threshold.
• Representative chromatograms should be provided with time scale and
attenuation indicated
Linearity / Range
• The working sample concentration and
samples tested for accuracy should be in the
linear range (concentrations Vs. Peak areas)
• Minimum 5 concentrations
• Dilute of stock solution or separate weighings
Linearity / Range
• Assay : 80-120% of the theoretical content of

active
• Content Uniformity: 70-130%
• Dissolution: ±20% of limits; eg if limits cover
from 20% to 90% l.c. (controlled release),
linearity should cover 0-110% of l.c.
• Impurities: reporting level to 120% of shelf
life limit
• Assay/Purity by a single method: reporting
level of the impurities to 120% of assay limit
Linearity / Range
Correlation coefficient (r)

API: ≥ 0.998
Impurities: ≥ 0.99
y-Intercept and slope should be indicated
together with plot of the data
Accuracy
Assay
API: against an RS of known purity, or via an alternate method of known
accuracy; analysis in triplicate.
FPP: samples/placeboes spiked with API, across the range of 80-120% of
the target concentration, 3 concentrations, in triplicate each.
Report per cent recovery (mean result and RSD): 100±2%
ICH Q2 states: accuracy may be inferred once precision, linearity and
specificity have been established. (Demonstration preferred).
Accuracy
Impurities: API/FPP spiked with known

impurities
Experienced in PQ:
Across the range of LOQ-150% of the target
concentration (shelf life limit), 3-5
concentrations, in triplicate each. (LOQ, 50%, 100%,
150%)
Per cent recovery: in general, within 80-120%,
depends on the level of limit
Precision
• System precision:
– by multiple injections (n ≥5) of a homogeneous
sample (standard solution).
– RSD ≤ 1% is recommended for assay;
– RSD ≤ 5% is recommended for related substances
(reference standards at the limit)
– Indicates the performance of the HPLC system
– As a system suitability test
Precision
• Repeatability (method precision)
– Multiple measurements of a sample by the same analyst
– A minimum of 6 determinations at the test concentration
(6 times of a single batch), or
– 3 levels (80%, 100%, 120%) , 3 repetitions each (combined
with accuracy)
– For Assay: RSD ≤ 2.0%
– For individual impurity above 0.05%, in general, RSD ≤ 10%
Precision
• Intermediate precision (part of ruggedness)
– Test a sample on multiple days, analysts,
equipments
– Repeat the method precision by different analyst
in different equipment using different lot of
column on different days
– RSD should be the same requirement as method
precision
• Reproducibility (inter-laboratory trial)
– Not requested in the submission
LOD/LOQ
• signal to noise ratio: LOD 3:1 , LOQ 10:1

– May vary with lamp aging, model/manufacturer of detector, column
• standard deviation of the response and the slope of the calibration curve
at levels approximating the LOD /LOQ
σ = the standard deviation of the response, base on

– the standard deviation of the blank
– The calibration curve
should be validated by analysis of samples at the limits.
LOD/LOQ
• LOD: below the reporting threshold

• LOQ: at or below the specified limit
Not required for assay/dissolution methods.
• Applicant should provide
– the method of determination
– the limits,
– chromotograms
Robustness
• The method's capability to remain unaffected by
small but deliberate variations in method
parameters
– Influence of variations of pH in a mobile phase
– Influence of variations in mobile phase composition
– Different columns (different lots and/or suppliers)
– Temperature
– Flow rate
• Evaluate the System suitability parameters
Robustness
Comparison of HPLC and GC
Sample Volatility Sample Polarity
HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase • PAH - inorganic ions
GC GC
• Samples are nonpolar
• Sample must be volatile
and polar
Sample Thermal Lability Sample Molecular Weight
HPLC HPLC
• Analysis can take place
• No theoretical upper limit
at or below room
temperature
• In practicality, solubility is
limit.
GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column
Sample Preparation Sample Size
HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase
GC GC
• Solvent must be volatile • Typically 1 - 5 mL

and generally lower
boiling than analytes
Separation Mechanism Detectors
HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)
GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds

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H : High

Separation analysis and/or preparation

Chromatogram containing three peaks

Reduction of the particle size of the stationary phase

- Thus pressure from 1000 to 5000 psi, pound per

Component A elutes the same time as a caffeine peak.

Component A is identified as caffeine.

Peak area (or height) is proportional to the concentration

The concentration of component A(caffeine) is determined by

Mobile phase elutes components.

I’ll need a separation

I have two separation techniques in my lab,

Normal phase Adsorption

Reversed phase Silica-

Size exclusion Porous

Ion exchange Ion exchange

Affinity Packings with

• Purification of sample amounts

•Purification for sample amounts < 1 g

• Most mobile compositions can be handled by

– Solvents must be miscible e.g. water/ethanol. An

Miscible with water, such as

B- Degassing using degasser.

C- Pre-saturation with the stationary phase in case

Example) methanol/water ( 60 : 40 v/v %)

The pH of the solvent (water) may be adjusted using

The selectivity of HPLC is affected by :

30% MeCN 45% MeOH

Fast Slow and better separation

• Water is “weak” solvent

30x0.4 cm C-18, 10 mm, 2

Snyder and Kirkland, Introduction to Modern

– Difference is in 2nd MeOH Syringols Guaiacols

(although greater retention 150

with MeOH of syringol is 5050

chromatography, pH should be O CH3

in range needed to produce

60:40 50:50 40:60

The higher the solvent viscosity, the harder it is for the

Where P = pressure drop in psi. F = flow rate (mL/min)

For water n = 1.0

For methanol n = 0.54

For 60% water n = 1.9

Gradient elution is divided

A = 80% Methanol, 20% Water

Method 1: A.CONC = 20%, B.CONC = 80%

• Low Pressure Gradient

For separation of a sample containing many components

Advantage of gradient elution method

Isocratic elution method Gradient elution method

Finepak SIL C18

Precautions in gradient elution method

- Can the gradient save time ?

Effect of temperature on retention time

The pumping system must...

Two groups of pumps:

Three types of pumps are available:

The solvent delivery system

• Filled with compressible liquid separated