HPLC

HPLC
I/II, Ist Semester M.Pharmacy
Dept . Of Pharmaceutical Analysis, JNTUH
Lecture by:

RAVI PRATAP PULLA

M.Pharm., Ph.D

Asso.Professor, SSJ College of Pharmacy, V.N.Pally,
Gandipet, Hyderabad-75.
SSJCP, Department of Pharmaceutical Analysis

1

HPLC – THE DEVELOPMENT OF A NAME
PERFORMANCE
PRESSURE
Price
Prestige
Peak
Profit
Propaganda
High

Promise

Chromatography

Philosophy
Polite
Problem
Ph (F) antasy

Liquid

Pragmatic
Pleasure
Passion


2

Introduction to Liquid
Chromatography
Columns
System Components
Applications
Troubleshooting


3

A Brief History of Chromatography
 1903: Russian botanist Mikhail Tswett separated
plant pigments
 1938: Russian scientists Izmailov and Shraiber use
“drop chromatography”.
 Later perfected as Thin Layer Chromatography
(TLC) by Kirchner in the U.S.
 1952: Martin and Synge receive Nobel Prize for
“invention of partition chromatography” or plate
theory to describe column efficiency.

4

 1966: HPLC was first named by
Horvath at Yale University but HPLC
didn’t “catch on” until the 1970s
 1978: W.C. Stills introduced “flash
chromatography”, where solvent is
forced through a packed column with
positive pressure.

5

Modern HPLC
 Late 1970s/early 1980s
►Instrumentation developed for high pressure solvent
delivery: pumps, autosamplers, diode array detectors
► More uniform packing material produced
columns

for

 Last 20 years
► Nothing really “new”, but by returning to the basic
theory of chromatography, even better columns are on the
market: smaller particle sizes which yield faster separations,
but require hardware to withstand higher pressures.

6

What is Chromatography?
 Separation of a mixture into individual
components.
 The separation uses a Column (stationary phase)
and Solvent (mobile phase).
 The components are separated from each other
based on differences in affinity for the mobile or
stationary phase.
 The goal of the separation is to have the best
RESOLUTION possible between components.

7

CHROMATOGRAPHY IS
INCOMPLETE WITHOUT
LEARNING
FEW BASIC TERMINOLOGIES
For any further clarification or details of the
below content(s) feel free to mail me :

ravipratappulla@gmail.com

8

Absorption

Bonded phase

Additive

Breakthrough volume

Adsorbent

Capillary column

Adsorption

Capillary LC

Adsorption isotherm

Cartridge column

Affinity chromatography

Cation exchange chromatography

Agarose

Channeling

Alumina

Chemisorption

Amphoteric ion-exchange phase

Chiral stationary phase

Analyte

Chlorosilane

Anion exchange chromatography

Co-ion

Bed volume

Column back pressure

BET (Brunauer, Emmet & Teller)
Column chromatography
method

Column plate number

Eluite

Column switching

Elute

Column volume

Elution

Competing base

Exclusion chromatography (Size)

Counterion

Extra column effects

Coverage

Fast protein LC (FPLC)

Cross-links

Frontal chromatography

Dead time (to / tm )
Displacement chromatography

Gel filtration chromatography
(GFC)
Gradient elution

Dynamic coating

Graphitized carbon packing

Effluent

Guard column

Eluate

Heart cutting

Eluent

Hold-up volume ( VM or tM )

Degassing


Hydrophobic interaction
chromatography (HIC)

Ion exclusion

Ion chromatography

Ion moderated partioning
chromatography (IMPC)

Imprinted phases

Ion pair chromatography (IPC)

Indirect detection

Linear chromatography

Injector (sample)

Linear velocity

Inlet

Liquid chromatography

In-line filter

Mobile phase velocity

Interparticle porosity (ee)

Open tubular column

Interstitial volume

Partition chromatography

Intraparticle porosity (ei)

Packed column

Intraparticle volume

Peak

Ion exchange chromatography

Peak area

Ion chromatography (IC)

Peak maximum


11

Peak width

Retention factor (k)

Phase ratio

Retention volume (VR or tR)

Plate height (H)

Separation factor (a)

Plate number (N)

Solid support

Pressure drop

Solute

Reduced mobile phase velocity (n) Stationary phase
Resolution (Peak) [ Rs ]/
Resolution(R)

Tailing

Reduced plate height (h)

Void volume

Relative Retention time (RRT)

Retention time (tR )

Interparticle time (tZ)

Capacity factor (k’)

Dead Volume(Vd)
Selectivity factor (α)

12

Activity

Adsorption chromatography

Asymmetry

Back pressure

Back flushing

Band spacing

Baseline

Baseline noise

Baseline resolved peak

Breakthrough volume

Buffer

Calibration standard

Capacity factor

Chain length

Channeling

Chromatogram

Chromatographic conditions

Chromatographic resolution

Chromatographic system

Column performance

Dead volume (Vm)

Dead time (tm)

Detection

Detector

Detection threshold

Detector linearity


13

Detector sensitivity

Differential Refractive Index( RI)

Electrochemical detector

Elution order

Elution chromatography

Eluotropic sequence

Elution volume

Extra column volume

External standard

Flow rate

Fluorescence detector

Frit

Fronting

HETP

Hydrophilic

Hydrophobic

Internal standard

Integrator

Interstitial particle volume

Ion exchanger

Ion suppression

Isocratic analysis

Isothermal chromatography

Ligand

Loading

matrix


14

Organic modifier

Overload

Partially resolved peaks

Particle size (medium)

Particle size distribution

Peak broadening

Peak area

Peak base

Peak height

Peak identification

Peak Quantitation

Peak shape

Phase system

Polarity

Pore diameter

Pore volume

Post column derivatization

Pre column

Pulsating flow

Recycling

Regeneration

Retention

Retention time

Retention volume

Sample

Sample capacity


15

Sample preparation

Separation capacity

silanization

Silanol groups

Sorbent

S.P chemically bonded

S.P

Surface modification

Specific surface

SFC( supercritical fluid
chromatography)

Vacancy chromatogram

Void

Void time


16

IUPAC RECOMMENDATIONS &
FREQUENTLY USED SYMBOLS IN
PARAMETER
SYMBOL
CHROMATOGRAPHY α
Separation factor
Selectivity factor (up to 1993 A.D)
Area

α
a/A

Diameter

de

Diffusion coefficient

d
ε / εt

Porosity
Flow rate (volumetric)

f

Plate height

h

Viscosity

η

Equilibrium distribution constant

k

Rate constant

k

Retention factor

k


17

PARAMETER
Capacity factor

SYMBOL
k’

Length of the column

l / L

Plate number /number of theoretical plates

n / N

Density

ρ

Pressure

p / P

Pressure (relative)

p

Radius

r

Temperature (absolute)
Time

t /T
t

Retention time

tr / tR

Velocity (linear)

u

Volume

v


18

PARAMETER

SYMBOL

Retention volume

vr

Mass (Weight)

w

Peak width

w

Difference

∆

Partial diameter

dp

Flow

F

Height equivalent of a theoretical plate(HETP)

H

Internal diameter of the column

I.D

Wavelength

λ

Iso electric point

pKa

Resolution

R

Death time

tm / t0


19

PARAMETER

SYMBOL

Gradient time

tG

Net retention time

tR'

Linear velocity

μ

Dead volume of apparatus

Vd

Pore volume

Vp

For any further clarification or details
of the above content(s) feel free to
mail me :

20

The Most Basic Explanation of
Chromatography Ever


21

 Drugs in multi component dosage forms,
analyzed by HPLC method because of the several
advantages like:
 Improved resolution of the separated substances
 Faster separation times
 The improved accuracy, precision, & sensitivity
with which the separated substances may be
quantified.

22

How Do You Get Separation?
 Hardware: pumps, injector, detector
 Column: particle diameter, column
size, packing materials
 Our seminar will focus on the
contribution of each factor to perform
separations.
23

 Column Considerations
► Theory (including, well...you know)
► Different Stationary Phases
 Hardware Components
► Pumps, Injectors, Detectors, etc.
► Examples of Application-Specific Configurations
 Applications
► Pharmaceuticals and Proteomics
► Food and Beverage, Environmental
► Research and Method Development

24

 System Troubleshooting
Leaks, Reproducibility, Column Care, and More
 Chromatography Software
Method and Sequence Setup
Calibration Curves and Reporting
 Chromatography Hardware
Modular LC-20 Prominence
Integrated LC-2010HT, Empower 2

25

Modern HPLC v/s
Traditional LC Methods
 Classical open-column LC.
 Thin-Layer Chromatography (TLC) and
paper chromatography.
 In modern HPLC the columns and packings
are, in general, highly refined, high in
resolving capacity, and are reusable.

26

HPLC and Pre-HPLC Techniques


27

MODES OF SEPARATION IN HPLC
 There are different modes of separation in HPLC:
►Normal phase mode
► Reversed phase mode
► RP - Ion pair chromatography
► Affinity/Bioaffinity chromatography
► Size exclusion chromatography
► Displacement chromatography

28

 Based on mode of chromatography
► Normal phase mode
► Reverse phase mode
 Based on principle of separation
► Adsorption chromatography
► Ion exchange chromatography
► Ion pair chromatography
► Size exclusion chromatography
► Affinity chromatography

29

 Based on elution technique
► Isocratic separation
► Gradient separation
 Based on the scale of operation
► Analytical HPLC
► Preparative HPLC
 Based on the type of analysis
► Qualitative analysis
► Quantitative analysis

30

COLUMN TYPES
Normal Phase LC
 Polar - stationary phase: Silica
 Nonpolar - mobile phase: Hexane, Ethyl acetate
 The LEAST polar compound comes out first
 Generally used for separation of non polar
compounds.

31

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

NOTE: The cyano column with a low polarity mobile phase (hydrocarbon with
a small amount of another solvent) will act as a normal phase column.


32

 this method separates analytes based on their affinity for
a polar stationary surface such as silica
 based on analyte ability to engage in polar interactions
(such as hydrogen-bonding or dipole-dipole type of
interactions) with the sorbent surface.
 Adsorption strengths increase with increased analyte
polarity
 interaction strength depends on the functional groups
present in the structure of the analyte molecule, but also
on steric factors

33

 more polar solvents in the mobile phase will decrease
the retention time of analytes
 hydrophobic solvents tend to induce slower elution
(increased retention times)
 traces of water in the mobile phase tend to adsorb to the
solid surface of the stationary phase forming a stationary
bound (water) layer which is considered to play an active
role in retention.
 governed
mechanism

almost

exclusively

by

an


adsorptive

34

Reversed-Phase LC
 Nonpolar - stationary phase: C8, C18
 Polar - mobile phase: Water, ACN, Methanol
 The MOST polar compound comes out first
 Generally used for separation of polar compounds


35

C18, C8

RP-HPLC Columns

:

Rugged, general purpose, highly retentive

C3, C4

:

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

NOTE

:

The cyano column with a high polarity mobile phase
(Water/MeOH) will act as a RP- Column.


36

 stationary phase is a silica which has been surfacemodified with RMe2SiCl, where R is a straight chain alkyl
group such as C18H37 or C8H17.
 retention time is longer for molecules which are less
polar, while polar molecules elute more readily
 can increase retention times by adding more water to
the mobile phase
 the affinity of the hydrophobic analyte for the
hydrophobic stationary phase stronger relative to the now
more hydrophilic mobile phase

37

 decrease retention time by adding more organic solvent
to the eluent
 RP-HPLC operates on the principle of hydrophobic
interactions
 RP-HPLC allows the measurement of these interactive
forces.
 The binding of the analyte to the stationary phase is
proportional to the contact surface area around the nonpolar segment of the analyte molecule upon association
with the ligand on the stationary phase.

38

 solvophobic effect is dominated by the force of water for

"cavity-reduction" around the analyte and the C18-chain versus
the complex of both.
 The retention can be decreased by adding a less polar solvent

(methanol, acetonitrile) into the mobile phase to reduce the
surface tension of water.
 Gradient elution uses this effect by automatically reducing

the polarity and the surface tension of the aqueous mobile
phase during the course of the analysis.
 Structural properties of the analyte molecule play an

important role in its retention characteristics.

39

 an analyte with a larger hydrophobic surface area (C-H,
C-C, and generally non-polar atomic bonds, such as S-S and
others) is retained longer because it is non-interacting with
the water structure.
 analytes with higher polar surface area (conferred by the
presence of polar groups, such as -OH, -NH2, COO– or
-NH3+ in their structure) are less retained as they are better
integrated into water.
 interactions are subject to steric effects in that very large
molecules may have only restricted access to the pores of
the stationary phase, where the interactions with surface
ligands (alkyl chains) take place.

40

 surface hindrance typically results in less retention.
 Retention time increases with hydrophobic (non-polar)
surface area.
 Branched chain compounds elute more rapidly than
their corresponding linear isomers because the overall
surface area is decreased.
 organic compounds with single C-C-bonds elute later
than those with a C=C or C-C-triple bond, as the double or
triple bond is shorter than a single C-C-bond.

41

 mobile phase surface tension (organizational strength
in eluent structure), other mobile phase modifiers can
affect analyte retention.
 entropy of the analyte-solvent interface is controlled
by surface tension, the addition of salts tend to increase
the retention time.
 mobile phase pH can change the hydrophobic
character of the analyte.
 For this reason most methods use a buffering agent,
such as sodium phosphate, to control the pH.

42

 Ammonium formate is commonly added in mass
spectrometry to improve detection of certain analytes by
the formation of analyte-ammonium adducts.
 volatile organic acid such as acetic acid, or formic acid,
is often added to the mobile phase if mass spectrometry is
used to analyze the column effluent.
 Trifluoroacetic acid is used infrequently in mass
spectrometry applications due to its persistence in the
detector and solvent delivery system, but can be effective
in improving retention of analytes such as carboxylic
acids in applications utilizing other detectors, as it is a
fairly strong organic acid.

43

 Reversed phase columns consist of alkyl derivatized silica

particles and should never be used with aqueous bases as
these will destroy the underlying silica particle.
 Can be used with aqueous acid, but the column should not

be exposed to the acid for too long, as it can corrode the
metal parts of the HPLC equipment.
 A good test for the metal content of a column is to inject a

sample which is a mixture of 2,2'- and 4,4'- bipyridine.
 Because the 2,2'-bipy can chelate the metal, the shape of

the peak for the 2,2'-bipy will be distorted (tailed)
when metal ions are present on the surface of the silica.

44

TYPICAL COLUMN SIZES


45


46

 Particle size: 5 µm, 3 µm, and smaller
 Mono dispersed 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
v/s higher carbon load

47

RP-HPLC MECHANISM






Synthesis of RP Packing
RP Column Properties
RP Retention Mechanisms
Important RP parameters
RP Optimization


48

Synthesis of RP Packing


49

RP COLUMN PREPARATION


50

COMMON RP PACKING


51

RP COLUMN PROPERTIES
► Hydrophobic Surface
► Particle Size and Shape
► Particle Size Distribution
► Porosity, Pore Size and Surface Area

52

PARTICLE SIZE
► Columns have a distribution of particle sizes

► Reported “particle diameter” is an average

► Broader distribution ---> broader peaks

53

Particle Size
Distribution of several column batches

Copyrights: Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82


54

RP MECHANISM (SIMPLE)


55

RP Mechanism (Advanced)
 Classical measures of retention
► capacity factors
► partition coefficients
► Van’t Hoff Plots
 Give bulk properties only
► do not give molecular view of
separation process

56

PROPOSED RP MECHANISMS
► Hydrophobic Theory
► Partition Theory
► Adsorption Theory

57

HYDROPHOBIC THEORY
 Chromatography of “cavities” in solvent created
by hydrophobic portion of analyte molecule
 Surface Tension
 Interaction of polar functions with solvent
 Stationary phase is passive

58

PARTITION THEORY
 Analyte distributes between aqueous mobile
phase and organic stationary phase
 Correlation between log P and retention
“organic” phase is attached on one end
 Does not explain shape selectivity effects

59

ADSORPTION THEORY
 Analytes “land” on surface - do not penetrate
 Non-polar interactions between analyte hydrophobic
portion and bonded phase
 Weak interactions
► dipole-dipole
► dipole-induced dipole
► induced dipole-induced dipole

60

None of the above can completely
explain all of the observed
retention in RP-HPLC


61

IMPORTANT REVERSED PHASE PARAMETERS

 Solvent (mobile phase ) Strength
 Choice of Solvent
 Mobile Phase pH
 Silanol Activity

62

SOLVENT STRENGTH
 Water is “weak” solvent
 Increased organic ---> decreased
retention
 Organic must be miscible with water

63

EFFECT OF SOLVENT


64

SOLVENT STRENGTH

COPYRIGHTS:Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286.


65

VARYING SELECTIVITY
30% MeCN

45% MeOH

70% Water

55% Water

30x0.46 cm C-18, 1.5
mL.min,254 nm, 10 mg each

COPYRIGHTS:Snyder and
Kirkland, introduction to
Modern Liquid
Chromatography, Wiley,
1979, p. 287.


66

pH
 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

67

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
COPYRIGHTS: Snyder and Kirkland,
Introduction to Modern Liquid Chromatography,
Wiley, 1979, p. 288.


68

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

69

Common buffers
Buffer

pKa Values

Phosphate

2, 7

Acetate

4.75

Citrate

3.08, 4.77, 6.40

Useful buffering between pH 2-8.


70

Silanol Activity
 RP ligands occupy about 50% of
silanols
 Others are “active”
 Weak acids

71

Silica Surface


72

Dealing with Residual Silanols
 Silanols cause peak tailing and excessive retention
 Endcapping
► bond a smaller group (helps a little)
 Pre-treatment of silica
► fully hydroxylated best
► high purity best

73

Silanol Interactions
 Hydrogen bonding
 Dipole-dipole
 Ion exchange
 Low pH --> silanols protonated
 Add basic modifier (TEA) to compete for sties

74

pH Effect on Tailing

Neue, p196


RP Optimization


76


77

IDEALIZED HPLC SEPARATION


78

VOID VOLUME
 The void volume is the amount of “dead” volume
in the column that is not taken up by the particles
of stationary phase.
 In general, there is approximately 0.1 mL of void
volume for each cm of column length, for columns
with a 4.6 mm i.d. and 5 µm particles

Vm ≈ 0.5dc2L
Where,

Vm is the column volume in mL,
L is the column length in cm, and
dc is the inner diameter in cm


79

 The void volume is exactly determined by
injecting a compound that is completely
unretained, then using the chromatogram to
calculate void volume.
 void volume = Elution time x flow rate


80

FACTORS INFLUENCING
RESOLUTION
 Capacity Factor, k’
 Selectivity Factor, α
 Efficiency, N


81

RESOLUTION
 For closely eluting or adjacent peaks, the
resolution equation may be expressed as:

Rs = 1 / 4[(α − 1) / α ] N [k ' /(1 + k ' )]
 The terms of capacity factor (k’), selectivity (α),
and efficiency (N) all contribute to resolution

82

THE RESOLUTION
EQUATION
 Resolution is defined as the completeness of
separation from one analyte to another
 In general, resolution may be expressed as:
Rs

= 2(Vrb - Vra)/(Wa + Wb)
= 2(trb - tra)/ (Wa + Wb)

Where,
Vra/b = retention volume of peak a/b
t a/b = retention time of peak a/b
SSJCP, rDepartment of Pharmaceutical Analysis

83

CAPACITY FACTOR, k

’

 The relative degree to which an analyte component is
delayed as it is eluted through a given system (retentivity).
k’ = (V r - V 0 )/V 0

=

(t r - t 0 )/t 0

Where,
Vr = peak retention volume
V0 = column void volume
tr = peak retention time
t0 = peak void time

 The larger the k’, the later the analyte elutes after the
void.

84

EFFECT OF k’ ON OVERALL RESOLUTION
 As k’ grows larger, its effect reaches a limit at a value of
about 10.
 Since k’ depends on retention time, longer columns
eventually have a diminished effect on resolution.


85

INFLUENCING THE CAPACITY FACTOR (k’)
 Retentivity (k’) decreases 2 - 3 fold for each 10% increase in mobile phase
strength.
 Which of these is easiest to change??
► Mobile Phase Strength As per the rule of thumb, altering the mobile phase strength also
alters the retention of the analytes.
► Bonded Phase Functionality (RP) As the bonded phase hydrophobicity increases (increasing alkyl
chain length, etc.) so will the retention of the analytes.
► Temperature As temperature increases, the retention time decreases. This does
not necessarily result in poorer separation because of the other
factors in the resolution equation.


86

Mobile Phase Strength v/s k
4.6 mm ID Column, 1 mL/min,
Changing MeOH % vs Water

0.079
100%
100%

90%
90%

’

Capacity Factor for Butyl Paraben (Peak 4)

0.212

0.472
80%
80%

70%
70%

1.127
2.813

60%
60%

7.666

50%
50%


87

Temperature Effect on k
50°C

’

2.1 mm ID Column, 0.35 mL/min, 50/50
MeOH/Water

45°C

40°C

35°C

30°C

25°C

20°C


88

Summary of k Effects
’

 A larger value of k’ means better resolution...to a
certain extent (k’ = 10 maximum)
 Increasing the mobile phase strength decreases k’
 Increasing the temperature decreases k’, but may
not result in a “bad” separation based on the other
factors affecting resolution.

89

Selectivity Factor, α

 The selectivity or separation factor represents the ratio of any two

adjacent k’ values, there by describing the relative separation of adjacent
peaks.

 This relationship is expressed as:
α = k’b/k’a

 If α = 1, two components are perfectly overlapping
 For early eluting peaks you want α to be large for good resolution.
 For later eluting peaks, α can be smaller and still have acceptable
separation.


90

Effect of α on Overall Resolution

 Remember the resolution equation?

Rs = 1 / 4[(α − 1) / α ] N [k ' /(1 + k ' )]
 Let’s only look at the part involving α

Rs = 1 / 4[(α −1) / α]

 And see how much resolution will improve with small changes in α


91

 For an α value of 1.1, the contribution of the
selectivity term is
(1.1 – 1) / 1.1 = 0.09
 For an α value of 1.4, the contribution of the
selectivity term is
(1.4 – 1) / 1.4 = 0.29
 So, a very small change in α leads to a more than
THREE-FOLD increase in the contribution to
resolution.

92

 As α grows larger, its effect reaches a limit at a value of about
5.
 Since α depends on components’ retention factor k’, longer
columns eventually have a diminished effect on resolution.


93

Influencing the Selectivity Factor α
 Which of these is easiest to change??
► Mobile Phase Type The importance of the type of interactions between
the
mobile phase and analytes is critical to the
optimization of the selectivity of a system.
► Column Type The bonded phase functionality can be selected by its
chemical nature to provide better selectivity in an
analytical method.
► Temperature Selective interactions between analyte molecules and
the stationary phase may not become evident until a
critical temperature is attained.

94

Summary of α Effects
 Since α is the ratio of two k’ values, the same general
statements apply:
►

Increasing the mobile phase strength decreases
individual
values of k’, but their ratio (α) may affect
resolution
► Increasing the temperature decreases individual values of

k’, but their ratio (α) may significantly affect resolution.

 A small increase in α leads to a large increase in resolution

95

Column Efficiency, N
 The column efficiency is defined as the degree to which
a column and/or other system components can physically
and chemically affect the separation of analytes.
 As column efficiency increases, analyte components will
elute in a smaller volume of the mobile phase, usually
observed as narrower or “sharper” peak shapes.
 Column efficiency is generally expressed in terms of
theoretical plate number.


96

Calculation of Theoretical Plates

N = A(tr /W)2
W

A
4

Wh

5.54

Wi

Method
Width measured at
Inflection point (60.7% of peak height)
½ Height

50% of peak height

W3s

3s

32.4% of peak height

W4s

16

4s

13.4% of peak height

W5s 25
Wb

9

5s

4.4% of peak height

16

Tangent Baseline, following tangent drawing

Constants A are different at each peak width, assuming a perfect Gaussian shape.
Real-world peaks often have tailing, so widths measured at the lower part of the peak more accurately reflect the
tailing when calculating N.


97

Calculation of Efficiency, N

Width measured at the
baseline after tangent
lines are drawn on the
peak.
Used when
tailing is minimal.

Width measured at
4.4% of peak height,
no tangents drawn.
Used when tailing is
significant.


98

Effect of N on Overall Resolution
 Do you STILL remember the resolution equation?

Rs let’s/lookαat − 1)part ]involving/(1 + k ' )]
= 1 4[( the / α N [k ' N
 Now
Rs = 1 / 4 N

 And see how much resolution will improve with
changes in N


99

 Since the contribution of N to resolution is a
square root, doubling N from 5000 to 10,000 only
increases the contribution to resolution by 41%.
 To double the effect on resolution coming from
N, we have to increase the value of N by a factor of 4
PLATE

Plates
5000
5,000
10,000
10,000
20,000
20,000

√N

CONTRIBUTION

141.4

100%

√N
Contribution
70.7 70.7
- - - - ----100 100
41% 41%
141.4
100%


100

Effect of N on Overall Resolution

 Note that there is no flattening of the curve like with k ’ and α.
 Resolution will continue to increase as theoretical plates increase.

101

Influencing the Efficiency, N
 Particle Size and Size Distribution The smaller the particle size and the narrower the range of the
particle size distribution, the more efficient the column.

 Packing Type Totally porous particles will also have greater efficiency than
solid or pellicular-shaped packing's, due to the additional
surface area attributable to the pores.

 Mobile Phase Viscosity As mobile phase viscosity increases, molecular movement
through the mobile phase is inhibited.

 Temperature For reverse phase chromatography, an increase in efficiency, N,
may be realized as column temperature is increased.

102

Effect of Particle Size on N
 Smaller particle sizes result in higher numbers of theoretical plates
Column
Diameter (mm)

Column Length
(cm)

Particle Size
(µm)

4σ Peak
Width (µL)

Theoretical Plates per
centimeter

10

25

10

1118

333

4.6

25

10

237

333

4.6

25

5

167

667

4.6

10

5

106

667

4.6

10

3

82

1111

4.6

3

3

45

1111

3

10

5

45

667

2

25

10

45

333

2

25

5

32

667

2

10

5

20

667

2

10

3

15

1111

1

25

10

11

333

1

25

5

8

667

1

25

3

6

1111

1

10

5

5

667

1

10

3

4

1111


103

Relative Influence of All Factors on Resolution
Parameter
Change

N

k’

α

Rs

Standard
+10% N
-25% N
-50% N
-60% N
-75% N
+10% k’
+10% α

10,000
11,000
7,500
5,000
4,000
2,500
10,000
10,000

2
2
2
2
2
2
2.2
2

1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.2

1.52
1.59
1.31
1.07
0.96
0.76
1.56
2.78

Note that changing α a very small amount has the biggest effect


104

Review of Factors

PARAMTER

INFLUENCED BY

TARGET VALUE

Efficiency, N

Column, system
flow path,
configuration

Minimum of 400
theoretical
plates /cm

Capacity factor,
k’

MP strength

1.0 - 10

Selectivity, α

M.P & S.P type

1.1 - 2

All of the above

1.3 – 1.5 or
greater

Resolution, Rs


105

Normal Phase v/s Reversed Phase
PARAMETER

NP

RP

Polarity of
packing

Medium to high

Low to medium

Polarity of
solvent

Low to medium

Medium to high

Elution sequence Low polarity first High polarity first
Increase solvent
polarity

Faster elution

Slower elution


106

ION EXCHANGE CHROMATOGRAPHY


107

 based on the attraction between solute ions and charged sites
bound to the stationary phase.
 The stationary phase contains ionic groups like NR⁺з, SO⁻з which
interact with the ionic groups of the sample molecules.
 This method is suitable for the separation of charged molecules
only.
 Solute ions of the same charge as the charged sites on the column
are excluded from binding
 solute ions of the opposite charge of the charged sites of the
column are retained on the column.
 Strong acids & basic compounds may be separated by RP mode by
forming ion pairs with suitable counter ions.

108

 Solute ions that are retained on the column can be
eluted from the column by changing the solvent conditions
 They include:
► increasing the ion effect of the solvent system
► by increasing the salt concentration of the
solution
► increasing the column temperature
► changing the pH of the solvent


109

 ion exchangers favor the binding of ions of higher charge
and smaller radius.
 increase in counter ion (with respect to the functional
groups in resins) concentration reduces the retention time.
 decrease in pH reduces the retention time in cation
exchange while an increase in pH reduces the retention time
in anion exchange.
 lowering the pH of the solvent in a cation exchange
column, more hydrogen ions are available to compete for
positions on the anionic stationary phase, thereby eluting
weakly bound cations.

110

TYPES OF ION EXCHANGERS
 Polystyrene resins
►These allow cross linkage which increases the
stability of the chain.
►Higher cross linkage reduces swerving,
which increases the equilibration time and
ultimately improves selectivity.
 Cellulose and dextran ion exchangers (gels)
► These possess larger pore sizes and low charge
densities making them suitable for protein
separation.
 Controlled-pore glass or porous silica

111

Examples
 Stationary phase contains charged groups
 SAX (Strong Anion Exchange): NH3+
 WAX (Weak Anion Exchange): NR2H+(DEAE)
[Di Ethyl Amino Ethanol]
 SCX (Strong Cation Exchange): SO3-

 WCX (Weak Cation Exchange): CarboxyMethyl
(CM)
 More highly charged analytes have stronger
retention
 More “bulky” stationary phases have weaker
retention

112

 IEC is widely used in the following applications:
►water purification
► preconcentration of trace components
► ligand-exchange chromatography
► ion-exchange chromatography of proteins
► high-pH anion-exchange chromatography
of carbohydrates and oligosaccharides

113

AFFINITY/ BIOAFFINITY CHROMATOGRAPHY


114

AFFINITY CHROMATOGRAPHY
 It uses highly specific biochemical interactions
for separations.
 The stationary phase contains specific groups
of molecules which can absorb the sample if
certain steric & charge related conditions are
satisfied.
 This technique can be used to isolate proteins,
enzymes, receptors , ligands as well as
antibodies from complex mixture.

115

Affinity chromatography can be used to:
 Purify and concentrate a substance from a
mixture into a buffering solution
 Reduce the amount of a substance in a mixture
 Discern what biological compounds bind to a
particular substance
 Purify and concentrate an enzyme solution.

116

Size Exclusion LC (or) Gel Permeation (or) Gel
filtration


117

 Stationary phase is a polymer (polystyrene-divinyl benzene or acrylamide) with
a defined pore size
 Large compounds cannot fit into the pores and elute first
 Used to determine molecular weight distribution of polymers
 Separates molecules according to their molecular mass.
 Largest molecules are eluted first and smaller molecules last.
 useful for determining the tertiary structure andquaternary structure of
purified proteins.
 used primarily for the analysis of large molecules such as proteins or polymers.
 SEC works by trapping these smaller molecules in the pores of a particle.
 widely used for the molecular weight determination of polysaccharides.


118

 larger molecules simply pass by the pores as they are too large to
enter the pores.
 Larger molecules therefore flow through the column quicker than
smaller molecules, that is, the smaller the molecule, the longer the
retention time.
 separates particles on the basis of molecular size (actually by a
particle's Stokes radius or Stokes-Einstein radius, or hydrodynamic
radius (RH).
 named after George Gabriel Stokes is the radius of a hard sphere
that diffuses at the same rate as the molecule.
 generally a low resolution chromatography and thus it is often
reserved for the final, "polishing" step of the purification.

 The main application of gel-filtration chromatography:
► fractionation of proteins and other water-soluble
polymers
► while gel permeation chromatography is used to
analyze the molecular weight distribution of organicsoluble polymers.
► Either technique should not be confused with gel
electrophoresis, where an electric field is used to "pull" or
"push" molecules through the gel depending on their
electrical charges.

120

DISPLACEMENT CHROMATOGRAPHY
 A molecule with a high affinity for the chromatography
matrix (the displacer) will compete effectively for binding sites,
and thus displace all molecules with lesser affinities
 displacement chromatography has advantages over elution
chromatography in that components are resolved into
consecutive zones of pure substances rather than “peaks”.
 because the process takes advantage of the nonlinearity of
the isotherms, a larger column feed can be separated on a given
column with the purified components recovered at
significantly higher concentration.

121

Aqueous Normal-Phase Chromatography
(ANP)
 ANP is a chromatographic technique which
encompasses the mobile phase region between RPC
and organic normal phase chromatography
(ONPC).
 This technique is used to achieve unique
selectivity for hydrophilic compounds, showing
normal phase elution using reversed-phase
solvents.

122

ISOCRATIC & GRADIENT ELUTION
 A separation in which the mobile phase composition remains
constant throughout the procedure is termed isocratic (constant
composition).
 Word was coined by Csaba Horvath
 A separation in which the mobile phase composition is changed
during the separation process is described as a gradient elution
 In isocratic elution, peak width increases with retention time
linearly
 leads to the disadvantage that late-eluting peaks get very flat and
broad.

123

 Gradient elution decreases the retention of the latereluting components so that they elute faster, giving
narrower (and taller) peaks for most components
 improves the peak shape for tailed peaks, as the
increasing concentration of the organic eluent pushes the
tailing part of a peak forward.
 increases the peak height (the peak looks "sharper")
 may include sudden "step" increases in the percentage
of the organic component, or different slopes at different
times.

124

 In isocratic elution, the selectivity does not change if the
column dimensions (length and inner diameter) change
 In gradient elution, the elution order may change as the
dimensions or flow rate change
 The driving force in RPC originates in the high order of
the water structure.
 The role of the organic component of the mobile phase is to
reduce this high order and thus reduce the retarding strength
of the aqueous component.

125

ISOCRATIC SYSTEM
 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

126

ISOCRATIC SEPARATION
 1 pump and premixing
 4.6 mm ID Column, 1 mL/min, Changing MeOH % vs
Water


127

 1 pump with valve and premixing

To Column
To Column

A = 80% Methanol, 20% Water
B = 70% Methanol, 30% Water
ABCD

C = 60% Methanol, 40% Water
D = 50% Methanol, 50% Water


128

 1 pump with mixer – let the pump do the work!

To Column
To Column

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



129

LOW PRESSURE GRADIENT
1 Pump, solvents are mixed before the pump
Requires degassing
To Column
To Column

ABCD


130

HIGH PRESSURE GRADIENT
Binary Gradient
2 Pumps and Mixer

Ternary Gradient
3 Pumps and Mixer
……….
……….
……….

……….
……….
……….

……….
……….
……….


131

HIGH v/s LOW PRESSURE GRADIENT
 High Pressure Gradient
► Multiple pumps are used with a mixer after the
pumps

 Low Pressure Gradient
► Solvents are mixed before the pump


132

Gradient v/s Isocratic Conditions: Summarized
 Isocratic
► mobile phase solvent composition remains constant with
time
► Best for simple separations
► Often used in quality control applications that support
and
are in close proximity to a manufacturing process
 Gradient
► mobile phase solvent (“B”) composition increases with
time
► Best for the analysis of complex samples
► Often used in method development for unknown mixtures
► Linear gradients are most popular (for example, the
“gradient” shown at right)

133

PRINCIPLE OF SEPARATION


The principle of separation is Adsorption.

 Separation of components takes
place
because of the difference
in affinity of
compounds towards stationary phase.

134

The principle of separation in normal phase mode and reverse
phase mode is adsorption.
 The component which has more affinity towards the adsorbent,
travels slower.
 The component which has less affinity towards the stationary
phase travels faster.
 Since no two components have the same affinity towards the
stationary phase, the components are separated.
1
Stronger
interaction

2

Weaker
interaction


135

PRESENT CHALLENGES
 Analysis of matrices like pharmaceutical dosage forms and
biological samples will always be challenging, due to their great
diversity, intricacy and complexity.
 Analyzing complex samples like biological products and
biological fluids is a significant challenge even with today’s
advanced and sophisticated instrumentation.
 Quality assurance & quality control of pharmaceuticals and
formulations play a vital role in ensuring the availability of safe
& effective drug products to the population.
 Quantitative estimation of the chemical entity of a drug
substance is pivotal to its quality assurance and control.

136

 The problem may be a simple one when one is dealing with a
pure and single substance.
 But, during the process of formulation, the original drug
substance of high purity is often diluted and mixed with other
additives.
 This may lead to interferences of the additives in the method of
estimation.
 The overall aim of our research is to develop new methods for
quantitative determination of novel drugs in pharmaceutical
dosage forms.
 The emphasis is to find new principles for separations using
liquid chromatography (HPLC) and to understand the mechanisms
behind.


137

INSTRUMENTATION


138

SCHEMATIC REPRESENTATION OF AN HPLC UNIT
1.Solvent reservoirs 2. Solvent degasser 3. Gradient valve
4. Mixing vessel for delivery of the mobile phase 5. Highpressure pump 6.Switching valve in "inject position” &
Switching valve in "load position” 7. Sample injection loop
8.Pre-column(guard column) 9. Analytical column 10. Detector
(i.e. IR, UV) 11. Data acquisition 12. Waste or fraction collector


139

BASIC FLOW CHART OF A HPLC SYSTEM SETUP


140

HPLC System Components
 Pumps

► Micro to Analytical to Preparative Flow Rates
► Isocratic and Gradient Configurations
 Degasser

► How it Affects Pumping and Sample Injection
 Valves

► Solvent Selection and Flow Selection

141

 Sample Injection
► Manual Injector or Autosampler
 Oven
► How Temperature Affects Separation
► Valves for Column Switching
 Detectors
► UV-VIS
► Diode Array
► Fluorescence
► Light Scattering
► Refractive Index
► Conductivity
► Mass Spectrometer
 Recorders and Integrators

142

 Fraction Collector
► Isolate Specific Sample Components
► Purify Compounds for Multi-Step
Synthesis
 Column
► Types of Packing Material
► Factors Affecting Separation
► Particle Size and Column Length
► Flow Rate and Temperature

143

A SOLVENT DELIVERY SYSTEM
 A mobile phase is pumped under pressure from one
or several reservoir and flows through the column at a
constant rate.
 For NP separation eluting power increases with
increasing polarity of the solvent but for reversed
phase separation, eluting power decreases with
increasing polarity.
 A degasser is needed to remove dissolved air and
other gases from the solvent.

144

HPLC DEGASSING
 Degassing removes dissolved air that interferes with check valve operation
 Refluxing
► not practicable
 Ultrasonic degassing
► ineffective & applicable for ACN/ Water
 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


145

Various solvent delivery systems


146

PUMP MODULES
Types:
 Isocratic pump
► delivers constant mobile phase composition;
► solvent must be pre-mixed;
► lowest cost pump
 Gradient pump
► delivers variable mobile phase composition;
► can be used to mix and deliver an isocratic mobile phase or a
gradient mobile phase
 Binary gradient pump
► delivers two solvents
 Quaternary gradient pump
► four solvents


147

 The pump is one of the most important component
of HPLC, since its performance directly affects
retention time, reproducibility and detector
sensitivity.
 Three main types of pumps are used in HPLC.
►Displacement pump
► Reciprocating pump
► Pneumatic (or) constant pressure pump

148

 DISPLACEMENT PUMP: It produce a flow that tends to
independent of viscosity and back pressure and also output
is pulse free but possesses limited capacity (250ml).
 RECIPROCATING PUMP: It has small internal volume (35400µl), their high output pressure(up to 10,000psi) and their
constant flow rates. But it produces a pulsed flow.
 PNEUMATIC (OR) CONSTANT PRESSURE PUMP:
► They are pulse free .
► Suffer from limited capacity as well as a dependence
of flow rate on solvent viscosity and column back
pressure.
► They are limited to pressure less than 2000 psi.

149

HPLC PUMPS – TWO BASIC TYPES
 Tandem piston
► Two pistons with different volumes (48 and 24 µL)
► During each stroke, 24 µL of liquid is delivered
► Best for higher analytical flow rates, up to 10 mL/min
► Some pulsation is observed, and pulse dampeners are
available
► Not recommended for pulse-sensitive detectors like
RID and CDD

150

TANDEM PISTON PUMP
Secondary
Piston ↓

← Primary Piston


151

DUAL PISTON
 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

152

DUAL PISTON


153

OTHER PUMP COMPONENTS
 Check Valves
► Control liquid movement in and out of the pump
head


154

 Piston/plunger seal
► Prevents solvent leakage out of pump head

 Inline filter
► Removes solvent particulates

155

VALVES USED WITH PUMPS
 Solvent Selection – 2 Solvents Per Pump
► Use for solvent switching


156

► Use for pump loading of large sample volumes


157

► Use for low pressure gradient formation


158

► Use for different gradients in method
development


159

SAMPLE INJECTION
SYSTEM
 There are three important ways of introducing
the sample in to the injection port.
► Loop injection : in which a fixed amount of
volume is introduced by making use of fixed
volume loop injector.
► Valve injection: in which, a variable volume is
introduced by making use of an injection valve.
► On column injection: in which, a variable
volume is introduced by means of a syringe
through a septum.


160


161

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 system


162

SAMPLE INJECTION – AUTOMATIC
 Fixed-Loop Auto sampler
► 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

163

Sample Injection… how is a sample actually put into an LC system

 Manual Injector:
1. User manually loads sample into the injector using a syringe
and then turns the handle to inject sample into the flowing mobile
phase which transports the sample into the beginning (head) of
the column, which is at high pressure

 Autosampler:
1. User loads vials filled with sample solution into the autosampler
tray (100 samples) and the autosampler automatically :
2. measures the appropriate sample volume,
3. injects the sample,
4. then flushes the injector to be ready for the next sample, etc.,
until all sample vials are processed for unattended automatic
operation

164

SAMPLE INJECTION – FIXED LOOP
 External syringe draws sample, then fills the
fixed-volume loop attached to the valve.


165

 Needle-in-the-flow path auto sampler
► 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


166

SAMPLE INJECTION TO FLOW PATH

Sample Loading


167

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
degassed!


MUST

be

168

%A
{H2O}

%B
%C
{MeOH}

Flow Rate Pressure
(mL/min)
(atmos.)

to column
load

Ready

inject

Rheodyne
Injector
Varian 9010 Solvent Delivery
System

to injector

through
pulse
dampener

Ternary
Pump
A

Column

through pump

C
B


from
solvent
reservoir

to
det
ect
or

169

CHROMATOGRAPHIC COLUMN
 The column is usually made up of heavy glass or stainless
steel tubule to withstand high pressure
 The columns are usually 10-30cm long and 4-10mm inside
diameter containing stationary phase at particle diameter of
25µm or less
 Column with internal diameter of 5mm give good results
because of compromise between efficiency, sample capacity,
and the amount of packaging and solvent required

170

 Within the Column is where separation occurs
 Key Point – Proper choice of column is critical for success in HPLC
 Types of columns in HPLC:
►Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 – 250
mm]
► Preparative (i.d. > 4.6 mm; lengths 50 – 250 mm)
► Capillary (i.d. 0.1 - 1.0 mm; various lengths)
► Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)
 Materials of construction for the tubing
► Stainless steel (the most popular; gives high pressure capabilities)
► Glass (mostly for biomolecules)
► PEEK polymer (biocompatible and chemically inert to most
solvents)

171

HPLC Columns Packing Materials
 Columns are packed with small diameter porous particles.
 The most popular sizes are: 5-μ m, 3.5- μ m and 1.8-μ m
 Columns are packed using high-pressure to ensure that they are stable
during use. Most users purchase pre-packed columns to use in their liquid
chromatographs

 These porous particles in the column usually have a chemically bonded
phase on their surface which interacts with the sample components to separate
them from one another for example, C18 is a popular bonded phase

 The process of retention of the sample components (often called analytes) is
determined by the choice of column packing and the selection of the mobile
phase to push the analytes through the packed column.


172

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

173

Why Use a Column Oven?
 Retention times decrease & higher flow rates
possible
2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water
50°C
45°C
40°C
35°C
30°C
25°C
20°C


174

DETECTORS
 The function of detector in HPLC is to monitor the mobile phase
as it merges from the column.
 Detectors are usually of two types:
► Bulk property detectors: It compares overall changes in a
physical property of the mobile phase with and without an
eluting solute e.g. refractive index ,dielectric constant or density.
► Solute property detectors: It responds to a physical property of
the solute which is not exbited by the pure mobile phase.e.g.UV
absorbance,fluoroscence or diffusion current.

171
175

TYPES OF DETECTORS
There are mainly 4 types of detectors are used in HPLC:
 Photometric detectors.
► Single wavelength detectors.
► Multi wavelength detectors.
► Variable wavelength detectors.
► Programmable detectors.
► Diode array detectors .
 Fluorescence detectors.
 Refractive index detectors.
 Electrochemical detectors.
 Evaporative light scattering detectors
 IR detectors
 UV detectors

176

PHOTOMETRIC DETECTORS
 These normally operate in the ultra violet
region of the spectrum .
 Most extensively used in pharmaceutical
analysis.

177

SINGLE WAVELENGTH DETECTORS

 Equipped with a low pressure mercury
discharge lamp.
 The absorbance is measured at the
wavelength of mercury at 254 nm.

178

MULTI WAVELENGTH DETECTORS
 Employ mercury and other discharge
sources.
 When used in combination with
interference filters allow a no of
monochromatic wavelengths to be
selected e.g. 206, 226, 280 , 313, 340 or 365
nm.

179

Multi-wavelength UV-Vis Absorption
Detector

Deuterium
Lamp


Photodiode
Array

180

VARIABLE WAVELENGTH
DETECTORS
 Use a deuterium light source.
 A grating monochromator to allow selection of
any wavelength in deuterium continuum
(190-360 nm).


177
181

UV-VISIBLE DETECTOR
 UV-Visible
► Wavelength range 190-700 nm
► D2 and W lamps
 Most common HPLC detector for a variety of samples
► Proteins and peptides
► Organic molecules
► Pharmaceuticals
 Monitor two wavelengths at one time

182

UV-Visible Detector


183

Variable wavelength detector


184

Variable UV/Vis Detector
ABS AUFS λ RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min
Ready


185

PROGRAMMABLE DETECTORS
 Allow the automatic change of
wavelength between and during the
chromatographic analysis.


186

DIODE ARRAY DETECTORS
 They are microprocessor – controlled
photodiode array spectrophotometers in
which light from an UV source passes
through the flow cell into a polychromator
which disperses the beam so that the full
spectrum falls on the array of diodes.


183
187

DIODE ARRAY DETECTOR
 Wavelength range 190-900 nm
 D2 and W lamps
 Spectral information about sample
 Create compound libraries to identify unknowns
 Monitor an entire wavelength range at one time – up to
790 wavelengths vs. only 2 with a UV detector

188

DIODE ARRAY DETECTOR


189

FLUORESCENCE DETECTOR
 These are essentially filter fluorimeter or
spectro -fluorimeters equipped with
grating monochromators, and micro flow
cell.
 Their sensitivity depends on the
fluorescence properties of the components
in the elute.

190

 Fluorescence detector
► Xenon lamp for light source
► Excitation wavelength range: 200-650 nm
► Emission wavelength range: up to 900 nm depending on
photomultiplier installed

 Used primarily for amino acid analysis
► Derivatize samples before (pre-column) or after
separation( post-column)

191

Fluorescence Detector


192

REFRACTIVE INDEX DETECTORS
 Which respond to the change in the bulk
property of the refractive index of the solution of
the component in the mobile solvent system.
 The sensitivity of the refractive index detector
is much less than that of specific solute property
detectors, they are useful for the detection of
substances(e.g ,carbohydrates & alcohols) which
do not exhibit other properties that can be used
as the basis for specific detection.

193

Refractive Index Detector
 For samples with little or no UV Absorption
 Alcohols, sugars, saccharides, fatty acids, polymers
 Best results when RI of samples is very different from RI of mobile
phase
 Flow cell is temperature controlled with a double insulated
heating block
 Requires isocratic separations
 Requires low pulsation pumps

194

RI BALANCE
 Fill sample and reference cell with mobile phase


195

RI ANALYZE
 Mobile phase flows through sample side only
 As the refractive index changes, the image on the photodiode is
deflected or “unbalanced”, and the difference in current to the photodiode
is measured.


196

Refractive Index Detector


197

ELECTROCHEMICAL DETECTORS
 These are based on standard electrochemical
principles involving amperometry,voltametryand
polarography.
 These detectors are very sensitive for substances
that are electroactive ,i.e. those that undergo
oxidation or reduction .
 They have found particular application in the assay
of low levels of endogenous catecholamines in
biological tissues,pesticides,tryptophan derivatives
and many drugs.

198

Electrochemical Detector


199

EVAPORATIVE LIGHT SCATTERING (ELSD)
 Also for low or no UV absorbing compounds
 Sometimes called a “Universal” detector
 Requires NO equilibration (unlike RID)
 Can be used with gradients and volatile buffers (unlike
RID)
 Semi-volatile compounds can be detected at low
temperatures

200

ELSD OPERATION
Column
Effluent

Nebulizer

Nebulizer Gas
(Air or Nitrogen)

Nebulization Chamber

Analyte

Drift Tube
(Heated Zone Evaporation Area)

PMT
Light Source

Amplifier
Light
Scattering
Cell


Signal
Output

201

ELSD v/s OTHER DETECTORS

 ELSD has higher sensitivity than UV and RID

 ELSD can be used with gradients, unlike RID


202

CONDUCTIVITY DETECTOR
 Flow cell contains 2 electrodes
 Measure ion amounts in sample
 REQUIRES low pulsation pumps
 Flow cell must be placed in a column oven

203

 Use in Environmental and water testing
► Fl-, Cl- NO3-, PO43-, SO42► Li+, Na+, K+, Mg2+, Cu2+, M-CN
complexes
 Determine organic acids in fruit juice
► Oxalic, Maleic, Malic, Succinic,
Citric
 Analyze surfactants
► Sulfonates, long/short chain
ammonium

204

Mass Spectrometer Detector
 Separate sample components as ions according to their mass to
charge (m/z) ratio
 Three stages to detection
 Vaporization: liquid from HPLC column converted to an aerosol
 Ionization: neutral molecules converted to charged species (either
positive or negative)
 Mass Analysis: filter ions by m/z ratio

205

TWO IONIZIZATION TYPES
 APCI: Atmospheric Pressure Chemical Ionization
► For molecules up to 1000 Da
► Singly charges ions
► Best for analysis of non-polar molecules

 ESI: Electrospray Ionization
► Can be used for large biopolymers
► Forms multiply charged ions
► Best for the analysis of polar molecules, especially
pharmaceutical products and proteins

206

MS DETECTOR
Heated
capillary

Q-array

Orthogonal source
geometry

Octapole

Electron Multiplier
Detector

Quadrupole mass analyser


207

FRACTION COLLECTOR
 Purify raw materials or compounds from
synthesis
 Collect by slope, level, time, volume
 Isolate single peaks per tube, or divide peaks
into small “slices” for extra purity


208

Temperature Control in HPLC: Why is it needed?
 Reproducibility
► Retention in HPLC is temperature-dependent
► If temperature varies, then it is difficult to assign “peaks” to specific
compounds in the chromatogram and the peak areas/heights may vary
 Solubility
► Certain chemical compounds may have low solubility in the HPLC
mobile phase
► If they are injected into the flow stream they may precipitate or other
difficulties may arise
 Stability
► Certain chemical compounds, especially biological compounds such as
enzymes or proteins, may not be stable at room temperature or higher
► The temperature needs to be much lower down to 4°C


209

How is Temperature Control Achieved?
 Three (3) ways the temperature of a column
could be controlled, use:
► Oven
► Heater Block
► Water bath

210

What is HPLC used for?
 Separation and analysis of non-volatile or
thermally-unstable compounds
 HPLC is optimum for the separation of chemical
and biological compounds that are non-volatile
 NOTE: If a compound is volatile (i.e. a gas,
fragrance, hydrocarbon in gasoline, etc.), gas
chromatography is a better separation technique.

211

 Typical non-volatile compounds are:
► Pharmaceuticals like aspirin, ibuprofen, or
acetaminophen (Tylenol)
► Salts like sodium chloride and potassium phosphate
► Proteins like egg white or blood protein
► Organic chemicals like polymers (e.g. polystyrene,
polyethylene)
► Heavy hydrocarbons like asphalt or motor oil
► Many natural products such as ginseng, herbal
medicines, plant extracts
► Thermally unstable compounds such as trinitrotoluene
(TNT), enzymes etc….

212

FOR QUALITATIVE ANALYSIS
 The identification(ID) of individual compounds
in the sample;
► the most common parameter for compound
ID is its retention time (the time it takes for
that specific compound to elute from the
column after injection);
►

depending on the detector used, compound
ID is also based on the chemical structure,
molecular weight or some other molecular
parameter.

213

FOR QUANTITATIVE ANALYSIS
 The measurement of the amount of a compound in a sample
(concentration); meaning, how much is there?

 There are two main ways to interpret a chromatogram (i.e.
perform quantification):

► determination of the peak height of a chromatographic peak
as measured from the baseline;

► determination of the peak area (see figure below);
 In order to make a quantitative assessment of the compound,
a sample with a known amount of the compound of interest is
injected and its peak height or peak area is measured.

 In many cases, there is a linear relationship between the
height or area and the amount of sample.

214

Preparation of Pure Compound(s)
 By collecting the chromatographic peaks at the exit of
the detector
 and concentrating the compound (analyte) by
removing/evaporating the solvent
 a pure substance can be prepared for later use (e.g.
organic synthesis, clinical studies, toxicology studies,
etc….).
 This methodology is called preparative chromatography.

215

Trace analysis
 A trace compound is a compound that is of interest to the
analyst but it’s concentration is very low, usually less than 1% by
weight, often parts per million (ppm) or lower;
 the determination of trace compounds is very important in
pharmaceutical, biological, toxicology, and environmental studies
since even a trace substance can be harmful or poisonous;
 in a chromatogram trace substances can be difficult to separate
or detect;
 high resolution separations and very sensitive detectors are
required

216


217

SEPARATION TECHNIQUES IN HPLC METHOD
DEVELOPMENT

GOAL

COMMENT

Resolution

Precise and rugged quantitative analysis
requires that Rs be greater than 1.5

Separation time

3-10 min is desirable for routine procedures

Quantitation

≤2% for assays; ≤ 5% for less-demanding
analyses; ≤ 15% for trace analyses

Peak Height

Narrow peaks are
signal/noise ratios

Solvent composition

Minimum mobile-phase use per run is
desirable

desirable


for

large

218

THE VALIDATION PROCESS
► It consists of four distinct steps:
Software validation
Hardware (instrumentation)
validation/qualification
Method validation
System suitability

219

HPLC SYSTEM QUALIFICATION


220

GOALS FOR AN IMPROVED ANALYTICAL
METHOD DEVELOPMENT
► Qualitative identification - structural information,
retention time, color change, pH etc
► Quantitative determination - accurate, precise and
reproducible in any laboratory settings
► Ease of use, viability to be automated, high sample
throughput, and rapid sample turnaround time.
► Decreased cost per analysis - using simple quality
assurance and quality control procedures

221

► Sample preparation minimizing - time, effort, materials,
and volume of sample consumed
► Direct output of qualitative or quantitative data evaluations, interpretation, printing out and transmission

OPTIMIZATION & ANALYTICAL FIGURES OF MERIT
► initial sets of conditions - resolution, peak shape, plate
counts, asymmetry, capacity, elution time, detection
limits
► quantifying the specific analyte of interest, accuracy and
precision of Quantitation and specificity must be defined.

222

► Chromatographic resolution adequate
► Limits of detections are lower
► Calibration plots are linear
► Sample throughout is increased
► Sample preparation before analysis is minimized
► Interference is minimized and identified
► Data acquisition - translated, interpreted, printed & stored
► Reproducibility of analytical figures of merit & Cost per analysis is
minimized


223

METHOD VALIDATION APPROACHES
► Samples of the given analyte
► Concentration in the matrix
► High degree of accuracy and precision
► Zero, Single and Double –Blind spiking methods
► Inter laboratory collaborative studies
► Comparison with a currently accepted compendium method

224

STEP-BY-STEP HPLC METHOD DEVELOPMENT, OPTIMIZATION
AND VALIDATION: AN OUTLINE
► Analyte Standard Characterization
► Method Requirements
► Literature Search and Prior Methodology
► Choosing a Method
► Instrument Setup and Initial Studies
► Optimization
► Demonstration of Analytical Figures of Merit with Standards

225

► Evaluation of Method Development with Actual Samples and
Derivation of Figures of Merit
► Validation of Figures of Merit
► Determination of Percent Recovery of Actual Sample and
Demonstration of Quantitative Sample Analysis
► Method Validation
► Preparation of Written Protocols and Procedures
► Transfer of Method Technology to Outside Laboratories and
Interlaboratory Collaborative Studies

226

► Comparison of Interlaboratory Collaborative Studies
► Preparation of Summary Report on Overall Method Validation
Results
► Summary Report of Final Method and Validation Procedures and
Results and also Preparation of Journal Article for Submission
THE OUTLINE PROTOCOL OF HPLC METHOD


227

STEPS FOR HPLC METHOD DEVELOPMENT
Information on sample, define
separation goals

Validate method for release to
routine laboratory

Need for special procedure
sample pretreatment, etc

Quantitative calibration

Choose detector and detector
settings

Choose LC method; preliminary
run; estimate the best
separation conditions

Check
for
problems
or
requirement
for
special
procedure

Optimize separation conditions


PARAMETERS USED IN METHOD VALIDATION


229

SPECIFICITY
► It is the ability to measure accurately and specifically the analyte
of interest in the presence of other components that may be
expected to be present in the sample matrix
► Specificity is also measured and documented in a separation by
the resolution, plate count (efficiency) and tailing factor
► Blank solution to show no interference with excipients or
degradation products or impurities
► Placebo to demonstrate the lack of interference from excipients
► Spiked samples to show that all known related substances are
resolved from each other

230

LINEARITY AND RANGE
► It is the ability of the method to elicit test results that are directly
proportional to analyte concentration within a given range
► Reported as the variance of the slope of the regression line
► ICH guidelines specify a minimum of five concentration levels
► Assay : 80-120% of the theoretical content of active
Content Uniformity: 70-130%
► Dissolution: ±20% of limits; e.g if limits cover from 20% to 90% l.c.
(controlled release), linearity should cover 0-110% of l.c.

231

► 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
► Correlation coefficient (r) = API: ≥ 0.998 & Impurities: ≥
0.99
► y-intercept and slope should be indicated together with
plot of the data


232

ACCURACY
► Measure of exactness of an analytical method or closeness of
agreement between the measured value and the value that is
accepted either as a conventional, true value or an accepted
reference value
► Measured as percentage of analyte recovered by assay, by spiking
samples in a blind study
► API (Active Pharmaceutical Ingredient): against an RS (Reference
Standard) of known purity, or via an alternate method of known
accuracy; analysis in triplicate
► FPP (Finished Pharmaceutical Product): samples/placeboes spiked
with API, across the range of 80-120% of the target concentration, 3
concentrations, in triplicate each

233

► Report % recovery (mean result and RSD): 100±2%
► Impurities: API/FPP spiked with known impurities
► Across the range of LOQ-150% of the target
concentration (shelf life limit), 3-5 concentrations, in
triplicate each. (LOQ, 50%, 100%, 150%)
► % recovery: in general, within 80-120%, depends on the
level of limit
► ICH Q2 states: accuracy may be inferred once precision,

linearity and specificity

234

LOD / LOQ
► LOD: the lowest concentration of an analyte in a sample that can
be detected though not necessarily quantitated.
► LOQ: the lowest concentration of an analyte in a sample that can
be determined with acceptable precision and accuracy under the
stated operational conditions of the method

► 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, based on the standard deviation of the blank & the calibration curve & S =
Slope


► should be validated by analysis of samples at the limits
► 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

236

ROBUSTNESS / RUGGEDNESS
► Robustness: capacity of a method to remain unaffected
by small deliberate variations in the method parameters
► Ruggedness: degree of reproducibility of the results
obtained under a variety of conditions, expressed as % RSD
► Evaluated by varying method parameters such as percent
organic solvent, pH, ionic strength or temperature ,
determining the effect on the results of the method,
columns, laboratories, analysts, instruments, reagents and
experimental periods.

237

SYSTEM SUITABILITY TESTING (SST)
► used to verify resolution, column efficiency, and
repeatability of the analysis system to ensure its adequacy
for performing the intended application on a daily basis.
►Parameters:
● Number of theoretical plates (efficiency)
● Capacity factor
● Separation (relative retention)
● Resolution
● Tailing factor
● Relative Standard Deviation (Precision)

238

Center for Drug Evaluation and Research (CDER) Limits for SST


239

CHARACTERISTICS TO BE VALIDATED IN HPLC
CHARACTERISTICS

ACCEPTANCE CRITERIA

Accuracy/trueness

Recovery 98-102% with 80, 100 &
120% spiked sample

Repeatability

RSD < 2%

Intermediate precision

RSD < 2%

Specificity/selectivity

No interference

Detection limit

S/N > 2 or 3

Quantitation limit

S/N > 10

Linearity

Correlation coefficient r > 0.999

Range

80 – 120%

Stability of sample solution

> 24 hours or > 12 hours


240

TYPICAL HPLC INSTRUMENT VERIFICATION
REPORT
TEST ITEM
USER LIMIT
ACTUAL LIMIT
DAD noise

< 5 X 10-5 AU

1 X 10-5 AU

Baseline drift

< 2x 10-3 AU/hour

1.5 X 10-4 AU/hour

DAD WL calibration

± 1 nm

± 1 nm

DAD linearity

1.5 AU

2.2 AU

Pump performance

< 0.3% RSD RT

0.15% RSD RT

Temperature
stability
± 0.15° C
(column heater)

± 0.15° C

Precision of peak area

0.09% RSD

0.5% RSD


241

METHOD VALIDATION PROTOCOL
1. On day 1, a linearity test over 5 levels for both the drug
substance (bulk) and dosage form is performed
2. Comparison of the results between the drug substance
and dosage form fulfills the accuracy requirement
3. At the end of day 1, 6 repetitions are performed at 100%
of the drug substance for repeatability
4. Steps 1 and 2 are repeated over 2 additional days for
intermediate precision
5. LOQ is evaluated by analyzing the drug substance over 5
levels, plus 6 repetitions for precision
6. Baseline noise is evaluated over 6 repetitions of blank
injections for the determination of LOD.

242

TROUBLE SHOOTING
(TIPS & FACTS)
ASK PULLA

For any further clarification or details of
the below content(s) feel free to mail me :


243

1.What is HPLC anyway?
2. How to become friendly with your HPLC equipment?
3. How to get started?
4. Which column do I have to install in the HPLC
instrument?
5. How do I prepare a mobile phase?
6. What is the requirement of equilibrating the system
before the advent of sample preparation.
7. What do I have to pay attention to before starting a
measurement?
8. How do I start working with the HPLC equipment?
9. What's the reason for quitting your HPLC system?

244

SIMPLE TESTS & DECISION CRITERIA
10.What does the name of a column tell us?
11. Is this C18 column the right choice for my sample?
12. Why are polar solutes well separated with one C18 column and
hardly at all with another?
13. How can I clean the RP Phase quickly?
14. How best do I degas my mobile phase?
15. Methanol or Acetonitrile? Best choice of solvent…..?
16. The pH of the mobile phase too high or too low. What can I do?
17.What is the right ionic strength of the buffer?
18.How to make sense of the dead volume of an isocratic apparatus?
19.Producing a gradient chromatogram – influence of
instrumentation?
20. Does the pump work correctly, precisely or accurately?

245

21. How to test an HPLC instrument and its modules?
22. Injections of solutes as an aqueous solutions?
23. What is the largest tolerable injection volume?
24 . How critical are the temperature changes?
25. How to choose HPLC equipment and a supplier?
26. Is the current method a robust one?

PROBLEMS & THEIR SOLUTIONS
27. Sample preparation – how critical are which mistakes?
28. Flushing of an HPLC equipment?
29. Dirt in the UV detection cell?
30. The lamp is new – what happened to the peak?
31. What are the causes of pressure changes or deviations?

246

32. Is the right or the left pump head defective?
33. Baseline noise and damping?
34. The retention times increase- is it the pump or the M.P ?
35. Which buffer is right for which pH?
36. An interesting alternative for the separation of acids & bases
with a buffer…..
37. What can be the reasons for a change in retention times?
38. I use up a lot of RP columns; what should I do?
39. Why does my NP system not work any more?
40. Chemical tailing at the presence of metal ions?
41. How to avoid memory effects?
42. How do the default values on my PC affect the resolution?

247

TIPS TO OPTIMIZE THE SEPARATION
43. Which is the right injection techniques to get sharper peaks?
44. My peaks appear too early – how can I move them in an RP
system to later retention times?
45. How can I increase the plate number?
46. Limit of detection: How can I see more?
47. How can I speed up a separation?
48. How can I optimize a separation?
49. Dead volume capacity, capacity factor, selectivity – how can I use
them in everyday life?
50. Which flow is optimal for me?
51. How can I optimize a gradient elution?
52. Separation of ionic solutes? What works out best –end capped
phases, inert phases, phosphate buffer or ion pairing reagents?

248

SITUATION/SYMPTOM/CAUSE
EQULIBRATION
53. SLOW COLUMN
 RP- Ion pairing long chain

54.VARYING / VARIABLE RETENTION TIMES








gradient insufficient column regeneration time
ion pairing insufficient equilibration time
isocratic insufficient equilibration time
irregular column equilibration time
Leak
change in M.P composition
air trapped in pump

249

 buffer capacity insufficient
 contamination buildup
 equilibration time insufficient for gradient run or changes in
isocratic M.P
 first few injections – active sites
 inconsistent online M.P mixing or delivery
 selective evaporation of M.P component
 varying column temperature
 check valve malfunctioning
 pump cavitations, phase collapse (de-wetting process)
 Column temperature fluctuations
 First few injections adsorption on active sites
 column overloading
 sample solvent incompatible with M.P

250

 Column problem
 improper M.P
 column aging

55. INCREASED RETENTION TIME










decreasing flow rate,
changing M.P composition,
loss of bonded S.P,
active sites on column packing
Low M.P flow rate
Column temperature low
Improper gradient setting
Column activity increasing
System not equilibrated

251







M.P removing water from LSC column
Incorrect M.P
Loss of bonded S.P
M.P composition changing
Active sites on silica packing

56. DECREASED RETENTION TIME








column overloaded with sample
increasing flow rates
loss of bonded S.P or base silica from column
column aging,
basic compounds – pH too low
High M.P flow rate
Column temperature high

252








Improper gradient
Incorrect M.P
Column activity decreased
System not equilibrated
Deactivation by strongly retained garbage
Too strong sample solvent

57. RETENTION BEYOND TOTAL PERMEATION
VOLUMES
 SEC – solute interaction with S.P.


253

58. LOSS OF RESOLUTION











M.P contaminated/deteriorated
Obstructed guard or analytical column
Column overload with sample
Degraded column
Column not fully equilibrated
Loss of S.P from the column
Dirty column
Loss of column liquid phase
Distorted column bed
Wrong column or M.P


254

SENSITIVITY
59. Lack of sensitivity
 auto sampler flow lines blocked
 detector attenuation set too high
 first few samples injections
 sample adsorption in injector sample loop or column
 injector sample loop under filled
 not enough sample injected
 peak signals are outside
 detector’s linear range
 sample losses during sample preparation
 sample losses on column peak too broad


255

BASELINE
60. Distribution At Void
 air bubbles in M.P
 positive-negative differences in RI of injection solvent & M.P

61. BASELINE DRIFT
 Column temperature fluctuations
 Non homogeneous M.P
 Contaminant or air buildup in detector, sample or reference cell
 Plugged outlet line after detector
 M.P mixing problem or change in flow rate
 Slow column equilibration when changing M.P
 M.P contaminated or deteriorated or not prepared from high
quality chemicals

256

 Strongly retained materials in sample can elute as very broad
peaks and appear to be a rising baseline
 Detector not set at absorbance maximum but at slope of curve
 M.P or sample vaporizing
 Failing detector source
 Detector problem
 Solvent immiscibility
 Contamination bleed in system
 Solvent demixing
 Slow change in pump output
 Partial plugging of injection port or sample valve or column inlet
by particulate matter
 Contaminated or bleed column
 Contamination in detector cell

257








Change in detector temperature
Malfunction of detector source
Contamination in solvent reservoir
Previous M.P not removed
Negative direction
Positive direction

62. BASELINE NOISE (REGULAR)








Air in M.P or detector cell or pump
Pump pulsations
Incomplete M.P mixing
Temperature effect
Other electronic equipment on same line
Leak or partial blockage of loop injector valve or detector lamp problem
Dirty flow cell


258

63. BASELINE NOISE (IRREGULAR)
 Leak
 M.P contaminated or deteriorated or prepared from low quality
materials
 Detector or recorder electronics
 Air trapped in system
 Air bubbles in detector
 Detector cell contaminated
 Weak detector lamp
 Column leaking silica or packing material or column packing
passing through detector
 Continuous detector lamp problem or dirty in the flow cell
 gradient or isocratic proportioning - lack of solvent mixing &
malfunctioning proportioning valves

259

 occasional sharp spikes,
 external electric interferences,
 periodic pump pulse,
 random contamination buildup,
 spikes – bubble in detector & column temperature higher than B.P
of solvent

RECOVERY
64. POOR SAMPLE RECOVERY
 absorption or adsorption of proteins
 adsorption on column packing
 absorption on tubing and other hardware components
 chemisorption on column packing
 hydrophobic interactions between S.P & biomolecules

260

 less than 90% yield for acidic compounds irreversible adsorption
on active sites
 less than 90% yield for basic compounds irreversible adsorption
on active sites

LEAKS
65. LEAKY FITTING






A loose fitting
Stripped fitting
Over tighten fitting
Dirty fitting
Mismatched part/fitting

261

66. LEAKS AT PUMP







Loose check valve
Mixer seal failure
Pump seal failure
Pressure transducer failure
Pulse damper failure
Proportioning valve failure

67. INJECTOR LEAKS







Rotor seal failure
Blocked loop
Loose injection port seal
Improper syringe needle diameter
Waste line siphoning
Waste line blockage

262

68. COLUMN

LEAKS

 Loose end fittings
 Column packing in ferrule
 Improper frit thickness
69. DETECTOR





LEAKS

Cell gasket failure
Cracked cell window
Leaky fittings
Blocked waste line

263

PROBLEMS DETECTED BY
SMELL, SIGHT & SOUND
70. SOLVENT SMELL
 Leak
 Spill
71. HOT SMELL
 Overheating
72. ABNORMAL METER READING
 Pressure abnormality
 Column oven
 Detector lamp failing
73. WARNING LAMP
 Pressure limits exceeded
 Other warning signals

264

HPLC

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