Modern

Thin-Layer Chromatography

Eike Reich and Anne Schibli

CAMAG-Laboratory
Muttenz, Switzerland
2

Table of content
1. Typical analytical tasks and solutions offered by HPTLC

2. Fundamental concepts

3. Method development

4. Standardization and Harmonization of HPTLC

5. Quantitative TLC

6. Method validation
1. Typical tasks and solutions
offered by HPTLC
1.1 For the botanical industry
1.2 For the pharmaceutical industry
1.3 Others
4

1.1 For the botanical industry

 Qualitative
ƒ Identification
ƒ Adulteration, falsification
 Semiquantitative
ƒ Selection of raw material
ƒ In-process control, stability tests
ƒ Batch-to-batch conformity
 Quantitative
ƒ Assay based on markers
 TLC literature methods
5

Qualitative aspects

 Identification - Definition of identity

 Natural variability, chemotypes

 Authenticity of materials (reference material)

 Purity, adulteration, falsification

6

HPTLC vs. botanical ID

Botanical ID HPTLC
 Highly trained personnel  Basic analytical skills
 Intact plant (parts) only  Universally applicable
ƒ Intact plant (parts)
ƒ Powder
ƒ Extract
 Single specimen  Also „pooled“ samples
 Name  Fingerprint
 Positive identity  Qualitative identity
 No change with time  Can track changes
7

Identification of Wuweizi (Schisandra sp.)

Schisandra chinensis or
Schisandra sphenanthera?

a) UV 254 nm
b) Sulfuric acid, white light
c) Sulfuric acid, UV 366 nm

1=Gomisin A, 2=Gomisin N, 3=Schisandrin, 4= S. chinensis,

5=S. sphenanthera, 6= Wuweizisu C, 7=γ-Schisandrin, 8= Deoxyschisandrin
8

Natural variability - Reishi mushrooms

Ganoderma lucidum fruiting body

Ganoderma lucidum mycelium

Ganoderma applanatum fruiting body

9

Chemotypes - Ashwaganda (Withania somnifera)

a) UV 254 nm
b) Sulfuric acid, white light
c) Sulfuric acid, UV 366 nm
Type 1
Type 2
Type 3
10

Authenticity of materials (reference material)

 Definition

 USP, Chin.Ph., Ph.Eur.

 Supplier

 Problems
 11

Purity of material - Echinacea

Root powder
E. purpurea root

Botanical drugs

?
E. angustifolia root
 12

Purity of material - Echinacea

 Where are the

differences?
 Chemical race or
adulterant?
 What and how
much is
acceptable?
13

Goldenseal and typical adulterants

1 and 11: Standards (hydrastinine, hydrastine, palmatine, berberine)

2: Rumex crispus 3: Mahonia aquafolium 4: Coptis chinensis
5: Xanthorrhiza sp. leaves 6: Hydrastis canadensis 7: Xanthorrhiza sp. steam
8: Xanthorrhiza root 9: Berberis nervosa 10: Berberis vulgaris
 14

Differentiation of Red Pepper and Paprika

Carotenoides
Flavonoids
Capsaicin
Paprika
Paprika Red Pepper
Hot Pk Red Pepper

Capsaicin
15

Semi-quantitative aspects

 Selection of raw material

 In-process control

 Stability tests

 Batch-to-batch conformity
 16

Selection of raw material - Chaste tree (diterpenes)

Croatia

L
C V
17

In-process control - Bearberry leaves

Uva ursi leaves

1 Crude drug
2 Aqueous extract
3 Finished product
4 References
5 Excipients

1 2 3 4 5
18

Stability tests

 Reproducibility of chromatographic result

 Comparability of measurement

 Points of attention

 When is material stable? Stress tests

19

Methodical problems ?
20

Methodical problems ?
 21

Control

Light

HCl

NH3

TFA

Diethyl amine

H2O2

Heat (diss. extract)

Heat (dry extract)

Air
Stability tests - Stress test on Chaste tree
22

Batch-to-batch consistency - Chaste tree

23

Quantitative aspects

 Quantitation based on marker substances

ƒ Definition of marker

ƒ Certified standards

ƒ Sufficient separation power

ƒ Selectivity of separation

ƒ Suitable instrumentation
24

Further requirements

 Stable and well-documented method

ƒ Reproducibility

ƒ Derivatization (?)

ƒ Detection

ƒ Confirmation of results
 25
Goldenseal -
Quantitation of alkaloids by video densitometry

3
2
1

S S S S S S S

Hydrastine 1 2 3
26

Hyperforin - Calibration curves

27

1.2 For the pharmaceutical industry

 R&D

 Identification of materials

 Purity of substances

 Sub-component analysis

 Content uniformity tests

 Cleaning validation
28

Impurities of Buproprion HCl

233 nm

0.5% impurity

0.3%

0.2%: limit for 3-

chlorobenzoic acid

0.1%: limit for unknown

impurities

Samples
29

Sub-component analysis
30

Sub-component analysis
31

CUT of Cinchocaine hydrochloride

 32

Cleaning validation

Filename:T20049 Track:1 to 19 Used files: peak data: PEPTIDES, analysis data: PE0020
8/AUG/2000 9/AUG/2000

600 Height Peptide fragment A

AU Perm.dev.range: 100 %
sdv: 2.5
a0 = -16.11
AU a1 = 757.00
400 a2 = 2.328E+002

350

300

250

200

150

100
Michaelis-Menten regression 2
50

0 0
0 10 20 30 40 50
70 60 0.0 986.0
mm [ng ]
CAMAG SOFTWARE (c) 1998 Version - CAMAG SOFTWARE (c) 1998 Version - 4.06
33

1.3 Other applications

 Food and beverages

 Cosmetics
 Polymers
 Environmental
 Toxicology
 Colors
34

Polymer additives - example 1

35

Polymer additives: example 2

36

Polymer additives: example 2 cont.

Used files: peak data: CIBA1 , analysis data: CIBA3 24/AUG/2000

13016.8 Area Chimassorb 81

Perm.dev.range: 0 %
sdv: 2.5
r = 0.9993
a0 = -846.72
a1 = 20261.75
a2 = -7.226E+003

Polynomial regression

0.0
0.0 0.3 1.2
[ug ]
CAMAG SOFTWARE (c) 1998 Version - 4.05
37

Publications on the subject

 American Herbal PharmacopoeiaTM, Post Office Box
5159, Santa Cruz, CA 95063, USA

 Reich E, Blatter A: HPTLC for the Analysis of Herbal

drugs, Herbal Drug Preparations and Herbal
Medicinal Products. In: Sherma J, Fried B (eds)
Handbook of Thin-Layer Chromatography 3rd edition,
Chapter 18. New York: Dekker; 2003

 Reich E, Blatter A: Modern TLC: A Key Technique for

Identification and Quality Control of Botanicals and
Dietary Supplements. INSIDE LABORATORY
MANAGEMENT, May/June 2004, 14-18
2. Fundamental concepts
39

2. Fundamental concepts

 General aspects

 Theoretical background

 Gas phase - chamber configurations

 Stationary phase

 Mobile phase
40

Advantages of thin-layer chromatography

 Fast and reliable (30 min / 36 samples)
 Fit for purpose (avoiding the overkill)
 Low cost per sample (solvent, analysis time)
 Flexibility (stat./mob. phase, detection)
 Suitable for complex mixtures (one-way plates)
 Easy documentation (electronic images)
 Fully compliant with GMP, 21CFR 11

Visual !
41

Other advantages

 Parallel analysis of samples and standards

 Few matrix problems, plate is use only once

 Selective optimization for specific components

 Flexible and multiple detection

 „Multi-dimensional“ result

 Off-line technique adds flexibility

42

HPLC versus HPTLC

 43

GC versus HPTLC
Detection of falsification - Sage oil

GC
44

How chromatography works

 Sample is introduced into chromatographic system

(stationary + mobile phase).

 Sample components are dissolved in the mobile

phase and transported while interacting with
stationary phase.

 Different affinities of components to stationary /

mobile phase cause different retention.
45

The TLC system

gas phase

S A M P L E

stationary phase mobile phase

 46

Factors influencing TLC

1
saturated 80% r.h.
0.9 N-chamber
unsaturated
0.8 N-chamber
normal
0.7 development "overrunning”
35o C of development
0.6 S-chamber pre-adsorption
50% r.h.
0.5

0.4 reference
other silica gel
0.3 10o C brands
20% r.h.
0.2

Effect of the "weightiest" influence factors in adsorption TLC

Reference: lipophilic substance with RF=0.5, unsaturated S-Chamber
Silica gel H (Merck), benzene, 50% r.h.
47

Capillary flow
ΔE = 2γVn / r E = energy
γ = surface tension
Vn = molar volume
r = pore radius

zf 2 =κ .t
zf = distance front-immersion line
κ = flow constant
t = developing time
48

Mobile phase flow

9
8

7
6
z f [cm]

2 Toluene, ethyl acetate (95:5)

TBME, methanol, ammonia (20:2:1)
1
Ethyl acetate, methanol, water, formic acid (50:2:3:6)
0
0 5 10 15 20 25 30

t [min]
 49

Developing distance
8 cm
Chamomile oil 7 cm

6 cm

5 cm

4 cm
Substance pair
3 cm at Rf 0.5
 50

Developing distance
4 cm 6 cm
3 cm 4 cm 5 cm 6 cm 7 cm 8 cm
51

Optimal developing distance

 5 - 7 cm for HPTLC (6 cm)

ƒ about 10 minutes
ƒ extend only for many components

 12 - 15 cm for TLC (12 cm)

ƒ about 30 minutes
ƒ extension does not improve result
52

Partition-/adsorption isotherm
CS fresh mobile phase
K2 = 2 Cm
2 K2 K1

K1 = 0,2
Cm CS
1 10

Cs K1
K= selectivity a =
Cm
K2
K partition coefficient
CS concentration in stationary phase
CM concentration in mobile phase
53

Reasons and solutions for tailing

 Overload: reduce amount of substance

 High activity of layer: saturation, pre-conditioning
 Reaction: change or modify layer
 Polar solvent residues: drying
 Convex isotherm: change phase system
 Dissociation: add buffer
 Changing substance: inert atmosphere
54

Reasons and solutions for leading

 Wet starting zones & weak developing solvent:

Æ dry starting zone, use stronger solvent

 Concave partition isotherm

Æ change phase system

55

Separation mechanisms in TLC

 Adsorption chromatography

 Partition chromatography

 Ion-exchange chromatography

 Complexation chromatography
 56

Separation mechanisms in TLC

Adsorption Partition Complexation
Functional groups/ Chain length/ Number / position of
Polarity Lipophily double bonds
SiOH RP AgNO3
Hydro
carbons

Ether C10 0 Db

Ester 1 Db
C15
Carbonyls 2 Db

Alcohols C20

Acids
57

The TLC chromatogram

Start Peak 1 Peak 2 Peak 3 Front
Absorption [AE]

zF

Migration distance [mm]

58

Rf -value/hRf-range
 Retardation factor or ratio to front
 Relative position of a zone on the
TLC/HPTLC-plate
b
Rf = <1
a A

a: migration distance of the mobile phase front a a

b: migration distance of the fraction b
b

hRf = Rf * 100
59

Rst -value
 Relative position of zone of interest compared to
standard

bi
R
st = b
st A

ref.
bi migration distance of zone of interest bi
bst
bst migration distance of standard
60

Separation - resolution

Rs= 2Δz / wb1+wb2

Rs= 1/4(α-1)(RFN)1/2(1-RF) wb2

a b c Δz
(RF2)[1-(RF1)] wb1
α= (RF1)[1-(RF2)] z1 z2 zf
N = zx / H RF
H = wb2 / 16zx z0
61

Effects of N, α, and Rf
62

The a-term: selectivity (α-1)

 Depends on partition coefficient of both substances

 Depends on chemical nature of:

ƒ Sample

ƒ Layer

ƒ Developing solvent

 Is not simply ΔRF

63

The b-term: layer quality (RFN)1/2

 N is number of theoretical plates over entire

separation distance

 Affects resolution only by square root

 Resolution is proportional to RF

 Calls for high RF

64

The c-term: (1-RF)

 Calls for low RF
 Extreme RF effect:
ƒ RF =1 yields Rs=0
ƒ RF =0 yields Rs=0
 b and c term:
ƒ RF must be optimized
ƒ Rs maximum at RF=0.3
 In Ns chamber RF<0.75
 65
Influence of the developing distance
and Rf-values on the resolution

2
1.9
1.8

1.7 1.6

Rf 0.1 1.4
1.5
Rf 0.2
Rf 0.3 1.2

Resolution
1.3
Resolution

Rf 0.4
1
1.1 Rf 0.5
Rf 0.6 0.8
0.9 Rf 0.7
0.6
Rf 0.8
0.7 Rf 0.9 0.4

0.5 0.2

0
0.3
0 0.2 0.4 0.6 0.8 1
0 1 2 3 4 5 6 7 8 9
De v e loping distance [cm]
Rf-values
66

Diffusion and separation number (SN)

The longer the migration distance the more diffuse are

the zones and the smaller is the separation number!
67

Performance of TLC - system: SN

How many spots fit on a plate at Rs = 1?

SN = (Zf’ / b0 + b1) - 1
b0 = extrapolated half width at RF = 0
b1 = extrapolated half width at RF = 1

HPTLC SNmax: 15 (6 cm)

TLC SNmax: 25 (15 cm)
AMD ’’SNmax~ 40’’ (8 cm)
68

The gas phase

 Affects:
ƒ Layer activity
ƒ RF
ƒ Separation
ƒ Mobile phase
 Depends on:
ƒ Chamber type
ƒ Conditioning
69

Chamber types - Nu ;Ns ;Su ;Ss

mobile phase stationary phase filter paper

70

Su - chamber

 Gas space <3mm

 No pre-conditioning

 Solvent de-mixing

 Multiple fronts

 Reproducible separation

 Comparable to dry column

71

Publications on the subject

 Geiss F: Fundamentals of Thin Layer

Chromatography. Heidelberg: Hüthig; 1987

 Reich E, Blatter A, Meier B: TLC fort he Analysis of

Herbal Drugs: A Critical Review of the Status and
Proposal for Improvements of Monographs.
Pharmeuropa 15.3 (2003), 424-430.
3. The TLC process
3.1 Sample application
3.2 Selection of chamber
3.3 Development techniques and modes
3.4 Stationary phase
3.5 Mobile phase
3.6 Derivatization
3.7 Detection
73

3.1 Sample application

 Suitable solvent and reproducible application

technique for contact application

 Reduce speed of application

 Dilute sample and apply larger volumes

 Focussing

 Apply bands instead of spots

74

Sample application, influence of solvent

75

Proper sample application

Contact Spray-on Contact Spray-on

 76

3.2 Selection of the chamber

Twin Trough Chamber
77

Horizontal Developing Chamber (HDC)

1: HPTLC plate (layer facing down), 2: counter plate,

3: reservoir for developing solvent, 4: glass wick,
5: lid, 6: central reservoir
 78
Effects of the chamber on the
separation of Schisandra

TTCprecond. TTCsat. TTCunsat. HDCsat. HDCunsat. HDCsandwich

HPTLC silica gel 60 F254, toluene - ethyl acetate - acetic acid (70 : 33 : 3)
Left: S. chinensis, right: S. sphenanthera
 79

Automatic Developing Chamber

ADC 2
ƒ Saturation
ƒ Pre-conditioning
ƒ Activity
ƒ Developing distance
ƒ Drying
80

3.3 Development techniques

 Single development: standard

 Circular / anti-circular: Rf
 Orthogonal (2D): complex mixtures, stability
 Multiple: complex matrix, difficult
mixtures
 Gradient: wide polarity range
 Forced flow: increased separation power
 Automatic: improved reproducibility
81

Single development
82

Circular and anti-circular development

Circular development Anti-circular development

83

Other development techniques

Capillary-techniques
R single
horizontal
vertical

circular anticircular
antiparallel

R multiple

repeated multi step multi step AMD

descending ascending step gradient

polar non polar

1 2 3 1 2 3 1 2 3 1 2 3
84

Other development techniques

Multiple development Forced flow techniques

OPLC RPC HPPLC

2 2

p p
1 1

Continuous development
Movable lid

6 5 4 3 2 1
85

Multiple development (same mobile phase)

First Second
development development

TLC

Single development
on a HPTLC plate
86

Multiple development (same mobile phase)

First Second
development development
1st development: hexane
87

2nd development: mobile phase

polar matrix
Removal of non-

1st development: very polar solvent

mobile phase)

2nd development: mobile phase, less polar

Removal of
polar matrix

Application
Multiple development (different

1st development: methanol

2nd development: mobile phase

Focusing of zones
88

AMD characteristics
 Focussing to yield sharp zones
 Zone profile almost independent of separation
distance
 Separation distance independent of matrix
 Polarity gradient freely programmable
 Substances can be of different polarity
 Separation number > 40 on 80 mm developing
distance
89

AMD 2

 Completely computer controlled

 Reproducible solvent composition

 Exact volume measurement for each step

 Precise control of developing distance

 Vacuum check prior to start

 Documentation of method and result

90

The AMD 2 - instrument

91

AMD 2 - schematic
92

The AMD - principle

Multi-step development:
Focussing

step 1 step 2 step 3 step 4 step 5 step ... step n

93

AMD - focussing effect

94

AMD 2
 95

AMD - example
Separation of Rhubarb extracts

Gradient in 10 steps: Methanol-dichloromethane (40:60) to (10:90) in 9 steps, 40 mm

developing distance, then 1 isocratic step methanol-dichloromethane (10:90) over 70 mm
developing distance
96

“Two - dimensional” separation

1. remove difficult matrix
1.
2. Separate sample

2.

180°

... with different solvents, plate turned about 180°

97

Two - dimensional development

1.

1. 2.

90°

Same or different developing solvent, plate turned 90°

98

3.4 The stationary phase

 Normal phase chromatography

ƒ (straight phase)

ƒ polar stationary phase + non polar mobile phase

 Reversed phase chromatography

ƒ non polar stationary phase + polar mobile phase

 Polarity of the layer:

ƒ Si > NH2 > CN/Diol > RP-2 > RP-8 > RP-18
99

Silica gel
 Surface area 300 - 600 m2 / g
 Pore size 40 - 80 Å (usually 60 Å)
 Binder
ƒ Gypsum, starch, hydroxymethyl cellulose poly(acrylic acid)
 Active structures on surface :
ƒ Silanol Si-OH
ƒ Siloxane Si -O - Si
ƒ Hydrogen bonded water (activity change)
 Specific interactions
ƒ Acid - base
ƒ Dipole - dipole
ƒ Induced dipoles
ƒ Dispersion forces
100

Impregnated silica gel

 Caffeine:
ƒ PAH
 Boric acid:
ƒ Sugars
 Silver nitrate:
ƒ Number and position of double bonds
 Sodium acetate:
ƒ Terpene in Gingko biloba
 101

Impregnation with sodium acetate

Gingko terpenes

No impregnation 4% sodium 10% sodium

acetate, 2 s. acetate, 20 s.
102

Stationary phases based on silica gel

Cu+ polar phase
O OH
O H
N[ O
chiral phases
] NH2
8 O Si O medium polar chemically
Si Si bonded phases
O O O
[ ]
CH3 8 Si Si O Si O OH
O OH
O O O
[ ] 5 Si Si
CH3 O O C N
Si O Si
CH3 CH3
]
3

non polar chemically

bonded phases
[

CH3
103

Synthesis of chemically bonded phases

 104

Abbreviations on packages
F fluorescence indicator
254 excitation wavelength for F
PSC preparative layer chromatography, thickness > 0,25 mm
R highly purified adsorbent
RP 2,8,18 reversed phase chain length / C-atoms 2,8,18
W water wettable layer
s acid stabile indicator
60 average pore diameter in Ångström (= 6 nm)
G gypsum as binder
H free of artificial binders
105

TLC or HPTLC ?
TLC HPTLC
Average particle size: 10 - 15 µm 5 - 7 µm
Particle size distribution: wide narrow
Layer thickness: 250 µm 100, 200 µm
Number of samples: max. 12 36 - 72
Separation distance: 100 - 150 mm 30 - 70 mm
Running time: 30 - 200 min 3 - 20 min
Solvent consumption: 50 ml 5 - 10 ml
Detection limit, absorbance: 100 - 1000 ng 10 - 100 ng
fluorescence: 1 - 100 ng 0.1 - 10 ng
106

Particle size distribution

Volume %

Particle size [μm]

 107

Comparison TLC-HPTLC
Flavonoids (Ph.Eur.4)
TLC plate 20 x 20 cm

HPTLC plate 20 x 10 cm
 108

Activity of the stationary phase

Relative humidity
 Depends on H2O on surface
ƒ Activation: removal of
water at 120oC
ƒ Conditioning: exposure to
a fixed relative humidity
 Affects
ƒ RF values:
high activity -low RF
ƒ Separation
 Must be controlled
Separation of polyphenyls with n- hexane
109

Effect of relative humidity

Polyphenyls on silica gel, detection at 265 nm, Vario chamber
1. m-quinquephenyl, 2. m-quaterphenyl, 3. m-terphenyl, 4. biphenyl
110

Controlling relative humidity

Mass % %relative saturated % relative
sulfuric acid humidity salt solution humidity
10 96 Pb(NO3)2 98

20 88 KBr 84

30 75 NaNO2 66

40 56 NaHSO4. H2O 52

50 35 KF 31

60 16 HCOOK 21

70 3 ZnCl2. 1.5 H2O 10

111

Pre-washing? Activation?
 Development with methanol
 Immersion in isopropanol

 Drying in oven at 120°C (20 min)

 Cooling to room temperature
 Conditioning

 Do not activate !
112

Pre-washing
113

Polar bonded phases - Diol

 Less effected by humidity

 Normal and reversed phase mode

 Most useful bonded phase in NP mode

 Similar in selectivity to silica

 Less retentive

 Hormones, steroids
114

Polar bonded phases - Aminopropyl

 In NP mode separation by type and number of functional

groups

 Acts as H-bond acid or base in NP mode

 In RP mode weak anion exchanger

 Gives fluorescent spots with sugars upon heating

 Carboxylic acid, phenols, nucleotides, vitamins, xanthine

derivatives, sugars
115

Polar bonded phases - Cyano

 Moderately polar

 In NP mode similar to deactivated silica gel

 High selectivity for polar heterocycles

 In RP mode used in ion-pair separation of acids

 Pharmaceutical preservatives
 116

Comparison of bonded phases

Separation of Hypericum – extract

Silica gel 60 Silica gel 60 DIOL Silica gel 60 NH2

Mobile phase: Ethyl acetate, dichloromethane, acetic

acid, formic acid, water (100:25:10:10:11)
117

Comparison of bonded phases

Separation of Taxol derivatives

Silica gel 60 Silica gel 60 NH2

Mobile phase: Chloroform, acetonitrile (4:1).

1: paclitaxel, 2: deacetylbaccatin, 3: dihydroacetylbaccatin
 118
Comparison of bonded phases
Mode switching
Normal phase: THF, toluene, formic acid, water (24:12:3:1.5)
Silica gel Diol Cyano RP18 W

Reversed phase: methanol, formic acid, water (5.5:1:4.5)

Cyano RP18 W

Vitexin, orientin, isovitexin,

isoorientin, chrysin, and 5 Passion
Flower samples
119

Switching the mode of separation - Cyano

Pregnelolone, testosterone, estriol, detection MnCl2/H2SO4, UV 366, normal chamber

100% hexane 100% acetone 100% water

120

Chiral phases
 Triacetyl cellulose
 Tribenzoyl cellulose
 Cyclodextrins bonded to silica gel
 Dinitrobenzoylamide (from amino phase)
 CHIR plate
ƒ C18 impregnated with Cu2+-hydroxyproline
 Enantiomers
ƒ Beta blockers, amino acids, lactons, dipeptides
121

Applications of reversed phases

 Non polar substances (lipids)
ƒ Fatty acids, carotenoids, steroids
 Polar substances
ƒ Basic and acidic drugs
 Substances labile on active layers
ƒ Vitamins, antibiotics, cannabinoids
 Insecticides, alkaloids, barbiturates, anti-oxidants,
analgesics, food dyes
122

Speciality of reversed phases

Separation of test dye mixture, toluene,
normal chamber, no saturation
123

3.5 The mobile phase - functions

 Dissolution of the sample

 Separation of sample and matrix

 Transport to optimal RF-range

 Determination of selectivity of separation

 Affects plate height (HETP) through viscosity

 Not identical to developing solvent (!)

124

Requirements for the developing solvent

 Simple composition

 Small polarity differences between components

 Non-toxic

 Defined ‘‘pure‘‘ quality

 No reactivity with sample

 Low viscosity

 ‘‘Optimal‘‘ volatility
125

Solvent strength - ε°

 Dimensionless number between -0.25 to 1.2

 Adsorption energy per area unit of solvent molecule on a

given adsorbent

 Independent of adsorbent activity

 Pentane defined to be = 0.00

126

Solvent strength (ε°) on silica gel

Pentane / Hexane 0.00 Ethyl acetate 0.48

Cyclohexane 0.04 Acetone 0.50
CCl4 0.11 Acetonitrile 0.60
Toluene 0.27 Dioxane 0.60
Isopropyl ether 0.28 Pyridine 0.70
Dichloromethane 0.30 Propanol 0.82
Chloroform 0.36 Methanol 0.95
Diethyl ether 0.43 Acetic acid >>1
Geiss, Fundamentals of TLC, Huethig 1987
 127

Solvent strength

Hexan 100 95 90 85 80 70 60 10
0 5 10 15 20 30 40 90 Ethylacetat
128

Solvent strength of binary mixtures

The 0.8

solvent 0.7

strength 0.6

0.5

of ε ° 0.4
n-Propyl chloride
Dichloromethane
Acetone

mixtures 0.3
Pyridine

is 0.2

not 0.1

additive! 0
0 20 40 60 80 100
% modifier in Pentane
129

Solvent strength of mixtures with pentane

% polar component in pentane
ε0 CS 2 i-PrCl Bz Et 2O CHCl 3 CH 2Cl 2 Acetone
0.00 0 0 0 0 0 0 0
0.05 18 8 3.5 4 2 1.5
0.10 48 19 16 9 5 4 1.5
0.15 100 34 28 15 9 8 3.5
0.20 52 49 25 15 13 6
0.25 77 83 38 25 22 9
0.30 55 40 34 13
0.35 81 65 54 19
0.40 100 84 28
0.45 42
0.50 61
0.55 92
130

Solvent strength and selectivity

Effect of selectivity at constant solvent strength
a) Hexane with 15% ethyl acetate
b) Hexane with 20% acetone
c) Hexane with 10% isopropanol
d) Chloroform neat
a b c d
131

Selectivity groups (Snyder)

I Aliphatic ethers, trialkylamines, trialkylphosphates
II Aliphatic alcohols
III Pyridine, THF, DMSO, DMF, diethylene glycol
IV Benzyl alcohol,ethylene glycol,acetic acid, formamide
V Dichloromethane, 1,2-dichloroethane
VI Ketones, esters, dioxane, nitriles
VII Aromatic (halogenated) hydrocarbons,nitro comp.
VIII Chloroform, water, fluoroalcohols, m- cresol
132

Selectivity triangle
H+-acceptor

II I xa

xd
III
IV
VIII VI
V
VII
H+- donor xn dipole
133

3.6 Why derivatization?

 Detection of non-absorbing substances

 Increase detectability

 Detection of all sample components

 Selective detection of certain substances

 Induction of fluorescence

 Pre-chromatographic changes
134

Derivatization - when?
pre-chromatographic
sample
D
sample prep
D
application D

development
D
post-chromatographic (elution)
D
measurement
135

Derivatization - how?

 In the gas phase

ƒ Iodine, HCl, ammonia, chlorine

 Spraying

 Dipping

 Thermal activation
136

Dipping or spraying?
Dipping Spraying

 Uniform coverage  Form of art

 Reproducible  Little consumption

 Background coloration  Flexible

 Tide marks  Poor reproducibility

 Large volumes  Fumes

137

Derivatization: Spraying or dipping?

Spraying Dichloroquinone Dipping
chloroimide reagent /
ammonia vapor
138

BioLumineX
139

BioLumineX
detection: bioluminescence Vibrio fischeri 06699

A B C D E F G H I np np np
detection: derivat. anisaldehyde / 366 nm

A - I : camomile oil np : 4-nitrophenol

140

3.7 Detection
 Visual / Images
ƒ Color
ƒ Fluorescence quenching
ƒ Fluorescence

 Densitometry
ƒ Absorption
ƒ Fluorescence
141

Documentation system
142

Multiple detection: example Valerian

1: V. officinalis ( auth.) 2: V. officinalis (CA, USA) 3: V. officinalis (Dutch)
4: Valeriana radix (PHH8) 5: V. sitchensis 6: V. wallichi (Ind. V.)
V=Valerenic acid
HCl – Acetic acid (vis)
1 2 3 4 V 5 6 1 2 3 4 V 5 6

HCl – Acetic acid (UV 366) Anisaldehyde

 143

Densitometry - Technical Solution

Entrance Lens
Lamp Selector
Monochromator Entry Slit
Monochromator Grating
Mirror

Slit Selector

Reference PM
Beam Splitter

Measuring PM
Scanning Object
144

Densitometric evaluation, UV-Vis spectra

145

Densitometric evaluation
All tracks, one WL
146

Densitometric evaluation
One track, 30 WL
3. Method development
148

3. Method development

 Analytical goal

 Practical approach
149

Optimizing a method

 Separation power

 Selectivity

 Matrix effects

 Detection limits

 Reproducibility

 Time consumption

 Costs
150

What is known?
 Number of components
 Chemical structures
 Molecular weights
 Solubility
 Type of matrix
 Concentration range
 Other: pKa ; UV spectrum, etc.
 NOTHING
151

Practical approaches

 Standard systems

 Spot test

 4-solvent-approach

 Prisma-model

 CAMAG - Optimization scheme

152

Standard systems (adsorption)

 Chloroform - methanol

 Chloroform - acetone

 Dichloromethane - methanol

 Ether - toluene

 Ether - hexane - acetic acid

 Ethyl acetate - methanol

 Ethyl acetate - toluene

153

Standard systems (partition)

 Chloroform - methanol - water

 Chloroform - methanol - water - ammonia

 Chloroform - methanol - water - acetic acid

 Butanol - acetic acid - water

 Butanol - pyridine - water

 Ethyl acetate - formic acid - water

154

Spot test: trial and error

 Dissolve sample in least polar solvent

 Apply several spots (auto sampler)

 Apply ~ 20 μL of various solvents onto center of spots

 Evaluate circular chromatograms

 Try solvent mixtures

155

Spot test: advantages and disadvantages

 Rapid and fully automated
 Number and type of solvents is flexible
 Information about
ƒ solvent strength
ƒ suitable selectivity

 Miniaturized ring chromatogram

 Unsaturated, open system
 Not systematic
156

Spot test: example 1

Buckthorn
157

Spot test: example 2

Lavender
158

The 4-solvent approach: step 1

Heptane / DCE / ACN / MTBE

neutral non-localizing localizing-dipole localizing- basic
Find solvent strength
ε° DCE/Hept ε° ACN/Hept MeOH/DCE
0.00 0 0.35 8
0.05 3.5 0.40 24
0.10 10 0.45 52
0.15 18 0.50 88
0.20 32 0.60 100
0.25 58 0.70 28
0.30 100 0.80 52
159

The 4-solvent approach: step2

At proper strength change selectivity
1) DCE/Heptane 1
2) MTBE/Heptane
3) ACN/Heptane
4) 1+2 (1:1)
5) 1+3 (1:1) 4 5
6) 2+3 (1:1) 7
7) 1+2+3 (1:1:1)
... (x:y:z)
2 6 3
160

The 4-solvent approach: example

161

The 4-solvent approach: Advantages / Disadvantages

 Works well

 Theory well understood

 Elaborate preparation

 Not applicable for polar compounds

162

The Prisma model: step 1

Select 8 - 10 neat solvents

diisopropyl ether I 2.4 dichloromethane V 3.1

MTBE I 2.7 dichloroethane V 3.5
diethyl ether I 2.8 ethyl acetate VI 4.4
2-propanol II 3.9 dioxane VI 4.8
ethanol II 4.3 acetone VI 5.1
methanol II 5.1 acetonitrile VI 5.8
THF III 4.0 toluene VII 2.4
pyridine III 5.3 benzene VII 2.7
acetic acid IV 6.0 chloroform VIII 4.1
formamide IV 9.6 water VIII 10.2
163

The Prisma model: step 2

 Adjust solvent strength to yield RF between 0.2 and

0.8 (0.3 !)

 Strong solvents are “diluted” with hexane

 Weak solvents are made stronger with:

water, acetic acid, diethylamine (small amounts)

164

The Prisma model: step 3

 Select the three “best” solvents

 Equalize solvent strength
 Calculations are based on P’ (see table)
 Dilution to the strength of the weakest solvent with
hexane

P’ = Σ Pi Ψ Ψ= volume increment of components

165

The Prisma model: step 3

166

The Prisma model: step 4

 Change selectivity
 Mix components of equal strength

Example: MTBE (2.7), DCM (3.1), chloroform (4.1)

MTBE neat
DCM diluted: 2.7/3.1= 87% (13% hexane)
Chloroform diluted: 2.7/4.1= 66% (34% hexane)

1:1:1 mix (P’=2.7): 33.3% MTBE + 29% DCM + 22% C + 15.7% hexane
167

The Prisma model: advantages / disadvantages

 Easy to use
 Rapid results
 Universally applicable

 Difficulties with polar samples

 Calculations can only give hints
 Effects of the gas phase are not included
168

The CAMAG - optimization scheme

169

Optimization of RP-systems
H+ - acceptor
NP-system RP-system
hexane for water for
“dilution” MeOH MTBE “dilution”

THF

ACN
CH2Cl2
CHCl3

H+- donor dipole

170

Solvent strength and selectivity

Solvent Solvent strength Selectivity

water 0.0 VIII

methanol 2.6 II
acetonitrile 3.2 VI
acetone 3.4 VI
dioxane 3.5 VI
ethanol 3.9 II
isopropanol 4.2 II
THF 4.5 III
171

Solvent strength for mixtures

Calculated as sum of the volume increments of the
components

Example:

80% methanol in water: S = (0.8 x 2.6) + (0.2 x 0.0) = 2.08

80% acetonitrile in water: S = (0.8 x 3.2) + (0.2 x 0.0) = 2.56
80% THF in water: S = (0.8 x 4.5) + (0.2 x 0.0) = 3.6

Strength of 80% MeOH = 65% ACN = 46% THF

172

The VARIO chamber

 6 solvents side by side

 Saturated / unsaturated

condition

 Pre-conditioning

 Activity control

 Reproducible results
173

Summary

 Optimization of the developing solvent is the best way

to improve a separation.

 First adjust solvent strength for optimal RF (0.3)

 Then change selectivity

 Fine-tune separation conditions

4. Quantitative evaluation
175

Methods of quantitation

 Densitometry
ƒ visible and UV light
ƒ with or without derivatization
ƒ very sensitive
 Digital images
ƒ substance must be visible
ƒ easy to use
176

Densitometry - Principle

 Sample absorbs / emits light

 Plate absorbs / reflects light

 Measurement of difference in optical response

between sample and plate

 Optical response of sample on plate is different from

response of sample in solution

177

Densitometry - Detection modes

Transmittance

 limited application

 substance must absorb visible light

 plate irregularities increase noise

 particularly useful for gels

178

Densitometry - Detection modes

Reflectance

 light is scattered and reflected by particles of the

layer

 sample absorbs portions of light

 described by Kubelka-Munk

 broad range of application

179

Densitometry - Detection modes

Fluorescence Quenching

 fluorescence indicator UV 254 or / and 366 nm

 emits green or blue light

 sample decreases emission

 less sensitive than reflectance

180

Densitometry - Detection modes

Fluorescence

 sample emits light after excitation by UV light

 most sensitive method (ng - pg)

 independent of sorbent reflectance

181

Densitometry - Problems

 Sample is not only on surface of plate

 Beer’s Law does not apply

 Calibration curves are usually not linear

 Signal influenced by plate

 Internal standard required

182

Quantitative determination

in solution on TLC-layers (reflection)

I0 Rd
I0
d
I R R0
d

I
Lambert-Beer Law Kubelka-Munk-equation
E=ε*c*d (1 - R)2
= 2.303 e C / S
E~c 2R
183

Quantitative determination on TLC-layers

Fluorescence equation

F = Φ I0 ε b c
F = signal
Φ = quantum efficiency
I0 = source intensity
ε = molar extinction coefficient
b = layer thickness
c = sample concentration
184

Fluorescence - Advantages

 Up to 100 times more sensitive than reflectance

 Linear range of 2 - 3 magnitudes

 Calibration curves are most often straight lines

through origin

 Integrated signal is independent of shape of spot

185

Fluorescence - Problems

 Limited application

 Quenching by atmospheric oxygen

 Matrix interference

 Emission maxima change with sorbent

 Often decreases with time

186

Fluorescence enhancement
 Avoid relaxation of sample
ƒ add enhancer : liquid paraffin, glycerol, triton X-
100
 Fine tune excitation wavelength
 Avoid matrix interference
ƒ enhancers will equalize response from sample and
standard
 Fluorogenic derivatization
187

Calibration Curves
 LINEAR y= a0 +a1x
ƒ low sample concentration
ƒ narrow calibration range
 POLYNOMIAL y= a0+a1x + a2x2
ƒ low sample concentration
ƒ wide calibration range
 MICHAELIS MENTEN y = a0 +(a1x/a2+x)
ƒ high sample concentration
ƒ wide calibration range
188

Linear calibration curve

189

Polynomial calibration curve

190

Michaelis-Menten calibration curve

191

Analysis of Australian red wine samples

Calibration for
B e n utz te D ate ie n : P emalic
akd ate n :acid
M A L IC _ AR , A n a ly se n d ate n : 1/M A R /20 00

Isopropyl
512 6.3 F laecether:
he formic acid: m alic acwater
id (85:15:5)
Z ul. Ab w eic h.: 0
sd v : 2.8
r = 0.9989
Standards a0 = -53 7.70 Wine Spiked samples
a1 = 57 0.2 3

Succinic acid
Lactic acid

L in eare R eg re ss io n
Malic 0.0
acid
0.0 2.0 10.0
[ug ]
C AM A G S O F T W A R E (c) 19 98 V ers io n -
192

Glycerin in tobacco
193

Acetylsalicylic acid derivatives

Acetylsalicylic acid
Salicyl amide

Salicylic acid

Acetylsalicylic acid
Salicyl amide
194

UV spectra

Acetylsalicylic acid
Salicyl amide

Salicylic acid
195

Calibration curves
196

Quantitation of sucralose and fructose

Beverage 1

Beverage 2
Sucralose

Fructose
Calibration for sucralose
Y=14.188 + 0.234*X r=1.00 sdv=0.08%
197

Aflatoxins in food matrix - corn flour

 198
Goldenseal -
Quantitation of alkaloids by video densitometry

3
2
1

S S S S S S S

Hydrastine 1 2 3
199

Publications on the subject

 Blatter A, Reich E: Qualitative and Quantitative
HPTLC Methods for Quality Control of Stephania
tetrandra, JLC & RT 27, No. 13, pp. 1–14, 2004

 Blatter A, Reich E: Analysis of Aristolochic Acids in

Chinese Drugs by High Performance Thin-Layer
Chromatography (HPTLC), JPC, in print
5. Standardization and
harmonization
201

5. Standardization and harmonization

 Chances and challenges
 SOP
ƒ Plate material
ƒ Application
ƒ Development
ƒ Derivatization
ƒ Documentation
ƒ Densitometry
 TLC in the Pharmacopoeias
202

Chances and challenges

 Comparability of results
 Optimization of output
 Increased reliability
 Improved international collaboration

 Agreement
 Compromises
 Communication
 Implementation
203

Is there a need for standardization?

Expert A Expert B

Fingerprint identification of Angelica and Levisticum

 204

Optimization and standardization

Angelica sinensis
(left tracks)
Levisticum officinalis
(right tracks)

Pre-washing No MeOH-CHCl3 (drying in oven 105° 30 min)

Chamber saturation 20 min (filter paper) 15 min (without paper)
Derivatization Dipping Spraying
Documentation Video camera Flat bed scanner
reflection and transmission
Activity of plate equilibrium with lab over night (P2O5)
humidity of 40% unknown humidity
205

Proposal for HPTLC SOP

 Plate material
ƒ pre-coated HPTLC plates, 20x10 / 10x10 cm
 Parameters for sample application
ƒ 8 mm bands, spray-on
 Preparation of developing solvents
ƒ measuring of volumes
ƒ storage
 Detailed description of development
ƒ 6 cm
 Derivatization
ƒ Dipping whenever possible
 Digital documentation
ƒ UV 254 nm
ƒ 366 nm
ƒ white light
 Plate labeling
ƒ Project number_year/month/day_plate number
206

TLC in the pharmacopoeias

 Revised Chapter 621 Chromatography

 State of the art HPTLC included

ƒ HPTLC plates

ƒ Instrumentation

ƒ Documentation

ƒ Update of quantitation
207

TLC in the pharmacopoeias

 State of the art methodology

 HPTLC

 Optimized, validated* methods

 Detailed description of results

 Color images of chromatograms

 Herbal drugs, finished products, adulterants

208

Accepted revisions
 Since 2005 ed. General Chapter (2.2.27)
ƒ Clear distinction between TLC and HPTLC
ƒ Parameters for sample application
ƒ Inclusion of derivatization devices
ƒ Inclusion of documentation
 New monographs
ƒ HPTLC plates in all herbal monographs
ƒ More specific description of individual steps

 Discussion about “Atlas”

209

Publication on the subject

 Reich E, Blatter A: A Standardized Approach to
Modern High Performance Thin-Layer
Chromatography (HPTLC). In: Vovk I, Medja A (eds.)
Proceedings of the International Symposium “Planar
Chromatography Today”, Novo Mesto: National
Institute of Chemistry; 2002

 Reich E, Blatter A, Meier B: TLC fort he Analysis of

Herbal Drugs: A Critical Review of the Status and
Proposal for Improvements of Monographs.
Pharmeuropa 15.3 (2003), 424-430.
 Laboratory

6. Method validation
211

6. Method validation

 Definition

 Pre-validation considerations

 Quantitative or qualitative method

 Parameters

 Linked to method development !

212

Definition – ICH

Validation is the formal proof that

the chosen analytical procedure is

suitable for the given task.

213

Pre-validation considerations

 Qualified instruments and equipment

 Defined, specified and tested solvents, reagents, plates
and standards
 Optimized and documented analytical procedure
(robustness check)
 A signed validation protocol (acceptance criteria,
statistical approaches)
214

Validation of qualitative methods

ICH guidelines are not comprehensive!

 Specificity (only required)

 Stability
 Precision
ƒ Repeatability
ƒ Intermediate precision
 Robustness
ƒ Plate
ƒ Humidity
ƒ Development
ƒ Derivatization
 Validated method
Robustness
Precision
Specificity
Flowchart for validation of

Validation protocol
qualitative methods

Stability
Optimization
215
Method selection
216

Stability

 Sample in solution (>3h)

 Sample on plate (>3h)
 2D-Separation
 Evaluate chromatogram immediately and
during 1 hour after derivatization or final
drying.
217

Stability of sample in solution / on the plate

Stability on the plate Stability in solution
over 3 h over 3 h

Stinging Nettle
218

Stability in the chromatographic system

2D-chromatography
1.

1. 2.

90°

... with the same mobile phase, plate turned 90°

 219

Stability in the chromatographic system

2D chromatography of Goldenseal
Chinese Ph. CAMAG USP
220

Stability in the chromatographic system

1 2

2D chromatogram of Angelica sinensis.

Left: formation of artifacts (arrows),
Right: the analyte is stable.
221

Stability of derivatization / result

Derivatization of of
Derivatization iridoids with with
diterpenes
dimethylaminobenzaldehyde
sulfuric acid reagent
reagent

Derivatization of flavonoids with

Natural Products reagent
 222

Stability – general acceptance criteria

 The analyte is stable

ƒ In solution not less than 3h

ƒ On the layer prior to development not less than 3h

ƒ During development

ƒ On the layer after development not less than 30 min

223

Validation protocol
 Description of experiments
 Definition of acceptance criteria

 Method for secondary lab (if applicable)

224

Specificity
Feverfew

1: Parthenolide
2: Feverfew (Tanacetum parthenium)
3: Mexican feverfew
4: Matricaria
5: Chamomilla romana
225

Precision
 Repeatability: 3 analytical solutions from individual
weightings of the same sample are analyzed in
triplicate on the same plate. The plate is repeated 3
times.
 Intermediate precision: same real sample is analyzed
on different days in the same laboratory.
 Reproducibility: Variability of results between different
labs.
226

Robustness
Applicability of the method under variable conditions,
with small changes or in different laboratories. Indicates
stability of method.
Chromatographic parameters
ƒ Relative humidity
ƒ Chamber type
ƒ Developing distance
Plate manufacturer
 227

Humidity / Activity
Green Tea polyphenols Test dye mixture
15% 30% 47% 60% RH 17% 47% 75% RH

Toluene, acetone, formic acid (4.5:4.5:1) Toluene neat

228

Chamber type
Ginseng species
TTC HDC
229

Plate manufacturer – St. John‘s Wort

Merck Si 60 Merck Si 60 Adsorbosil Uniplate
extra thin

Whatman M&N Merck Si 60 Uniplate G

without F
230

Validation of quantitative methods

Follow ICH guidelines
 Accuracy
 Precision
ƒ Repeatability
ƒ Intermediate precision
 Specificity
 LOD / LOQ
 Linearity / Range
 (Robustness)
231

During method development

 Separation of all sub components

 Achieving detection limits
 Evaluation of important factors (robustness)
ƒ relative humidity
ƒ activity
ƒ chamber type and - saturation
ƒ reproducibility of chromatography
232

Specificity for identification

 Sample, reference standard(s), pure and spiked
placebo are chromatographed side by side.

 Acceptance criteria
ƒ Adequate separation of the API from all other
components of the sample.
ƒ Fingerprints similar with respect to number, color
and sequence (different for adulterant, no
interference of matrix)

 Documentation can be performed using photographs

or video prints. The validation report must contain
these documents in original or digitized form.
233

Specificity - matrix
1-6: Standards
7, 8: Raw material
9: Extract
10: Tablet
11: Tablet matrix
234

Specificity for known impurities

Blank

100% Mazipredon, spiked

100% Mazipredon

1% Mazipredon + impurities

1% Impurities
235

Specificity for unknown impurities

Stress test: 1: Famotidin, 2: UV, 3: O2, 4: Base, 5: Acid, 6: Blank

6
5

2
1
236

Linear range (working range)

 Often non-linear calibration function
 Pseudo-linear range with at least 3 points
 For non-linear function at least 4 points
 Linear working range 10-point calibration with at least
8 independent and equidistant concentrations.
Highest and lowest concentration are determined in
duplicate.
 5 point calibration with 5 replicates at low and high
end of range
237

Linear calibration for hyperforin

5 concentration Highest concentration

levels 5 applications
2 applications

2 applications

2 applications

Lowest concentration
5 applications
238

Linearity test

r= 0.9966

300

200

100

0 500 1000 1500 2000

Residuals

-100

-200

-300

-400

-500
239

Linearity test

r= 0.9973

200

100

0 200 400 600 800

Residuals

-100

-200

-300

-400
240

Homogeneity of variance (F-test) - linearity

241

Precision
 Repeatability: same real sample is analyzed on the
same plate in 6 analytical solutions from individual
weightings. Information about variation in sample
preparation and application.
 Intermediate precision: same real sample is analyzed
on different days by different analysts using different
instruments and chemicals in the same laboratory.
Information about experimental and environmental
effects.
 Reproducibility: Variability of results between different
labs. Is not required.
242

Limit of quantitation (LOQ)

Concentration that allows quantitative detection and
discrimination from zero with statistical significance. (= 2-
3 time limit of detection [concentration that allows
detection of substance, 3 time the noise level])

ƒ can be approximated from chromatogram and

integrated baseline noise or graphically from
calibration function
243

Limit of detection (LOD)

 LOD is determined by applying and developing decreasing
quantities of API in duplicate. Smallest amount visible to
three analysts is LOD.

 Acceptance criterion:
ƒ Identification and/or qualification of all impurities >
0.1 %.
ƒ Validated LOQ < 0.05 % (50 % of specified limits).
 For semi-quantitative procedures no acceptance criteria
for LOQ are defined
ƒ LOD should be 10 – 20 % of specification.

 Insufficient LOD may be improved by either increasing

applied quantity or by using selective pre- or post-
chromatographic derivatization.
244

LOD, LOQ - graphical determination

245

Accuracy
Deviation of measured value from true value
 Systematic error
ƒ (calibration curve: intercept with y-axis, should be
close to 0)
 Recovery function with and without matrix
ƒ compare slope and intercept
ƒ F-test of residual variances
 Recovery rate
ƒ 85-115% (correction factor)
246

Accuracy
 Triplicate analysis of solutions of the analyte spiked
with 3 different concentrations of available impurities.
Recovery is calculated as percentace value of added
amount.
 Acceptance criteria:

n=3 3 concentrations Recovery RSD of recoveries

Impurity level </= 0.5 % 80 – 120 % </= 10 %

Impurity level >/= 0.5 % 90 – 110 % </= 5 %

247
Example: Quantitation
of tetrandrine in Stephania

Tetrandrine at
210 nm
248
Example: Quantitation
of tetrandrine in Stephania

Specificity: UV Spectrum
Tetrandrine standard
Linearity: 50 – 112.5 ng absolute
y = -4.544 + 1.339x
Tetrandrine in extract
r = 0.9996

Range: 60 – 90 ng absolute (target 75 ng)

Accuracy: not performed
Precision: 6 appl.: 0.45%, 3 plates: 1.2%
249

Publication on the subject

 Koll K, Veit M, Reich E, Blatter A: Novel approach to
validation of qualitative HPTLC methods for herbal
drugs, drug preparations, and herbal medicinal products
(HMP’s). Journal of AOAC International, May/June
2004, 909-912
 ICH Guideline Q2A
Validation of Analytical Procedures: Definition and
Terminology
 ICH Guideline Q2B
Validation of Analytical Procedures: Methodology
 Validation and Quality Assurance of Planar
Chromatographic Procedures in Pharmaceutical
Analysis K. Ferenczi-Fodor, Z. Vegh, A. Nagy-Turak, B.
Renger, M. Zeller; JAOAC 4 (84) 2001, p.1265 ff.