High performance

liquid chromatography

Anlytical HPLC: Seminars in Brazil, April 16 and

17, 2012 in Rio and Sao Paulo
Domingo Sanchez, PhD

The Presentation
Challenges in Analytical HPLC
Most common phases and their use
How to do it right and how to avoid mistakes
Trouble shooting
Practical examples
About Kromasil

Challenges in analytical HPLC

Sustainability
p SFC
p Subcritical water (RP)

Possibility to improve/change the analytical method after

submission to authorities
Improve selectivity/performance of columns by new
phases/smaller particles
Develop stationary with same properties for
analytical/preparative HPLC but with different particles
sizes

Trend towards smaller particles

Column L

Particle size

Flow rate

Rel. time

Rel. Rs

Rel. P

250 mm

10 m

0.5 ml/min

125 mm

5 m

1.0 ml/min

0.25

87.5 mm

3.5 m

1.43 ml/min

0.125

62.5 mm

2.5 m

2.0 ml/min

0.0625

16

45 mm

1.8 m

2.8 ml/min

0.0321

32

Most common phases and their use (1)

Reverse Phase, RP (Peptides, proteins)
p 60-70% of all separations
p C4, C8, C18, Phenyl

Normal Phase, NP (Synthetic mol., Natural Products)

p SIL, DIOL, AMINO, CYANO, HILIC

Chiral Phases, CSP (Optical Isomers)

p Polyssaccharide coated or inmobilize on silica
p Other

Supercritic Fluid Chromatograpy, SFC (As NP, New appl.

p DPA, SIL, DIOL, CYANO

Most common phases and their use (2)

Ion Exchange Phases, IEX (Peptides, Proteins)
p Polymeric, but silica based are increasing

Silica/Polymer (Composite) (For high pH separations)

p Eternity, Waters

For a complete list see USP Chromatography Column

Classification

Types of columns according to USP (1)

USP Chromatography Column Classifications
L1
L2
L3
L4
L5
L6
L7
L8
L9
L10
L11
L12
L13
L14
L15

C18 Octadecyl silane chemically bonded to porous silica or ceramic microparticles, 5 to 10 in

diameter.
C18 Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has
been bonded to a solid spherical core, 30 to 50 in diameter.
Porous silica microparticles, 5 to 10 in diameter.
Silica gel of controlled surface porosity bonded to a solid spherical core, 30 to 50 in diameter.
Alumina of controlled surface porosity bonded to a solid spherical core, 30 to 50 in diameter.
Strong cation-exchange packing-sulfonated fluorocarbon polymer coated on a solid spherical
core, 30 to 50 in diameter.
C8 Octyl silane chemically bonded to totally porous microsilica particles, 5 to 10 in diameter.
NH2 an essentially monomolecular layer of aminopropylsilane chemically bonded to totally
porous silica gel support, 10 in diameter.
SCX 10 irregular totally porous silica gel having a chemically bonded, strongly acidic cationexchange coating.
CN nitrile groups chemically bonded to porous silica microparticles, 5 to 10 in diameter.
Phenyl groups chemically bonded to porous silica microparticles, 5 to 10 in diameter.
A strong anion-exchange packing made by chemically bonding a quartenary amine to a solid
silica spherical core, 30 to 50 m in diameter
Trimethylsilane chemically bonded to porous silica particles, 3 to 10 m in diameter
Silica gel 10 m in diameter having a chemicallly bonded, strongly basic quaternary ammonium
anion-exchange coating
Hexylsilane chemically bonded to totally porous silica particles, 3 to 10 m in diameter

Types of columns according to USP (2)

USP Chromatography Column Classifications
L16
L17
L18
L19
L20
L21
L22
L23
L24
L25

L26
L27
L28
L29

Dimethyl silane chemically bonded to totally porous silica particles, 5 to 10 in diameter.

Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene
copolymer in the hydrogen form, 11 in diameter.
Amino and cyano groups chemically bonded to porous silica particles, 5 to 10 in diameter.
Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene
copolymer in the calcium form, 9 in diameter.
Dihydroxypropane groups chemically bonded to porous silica particles, 5 to 10 in diameter.
A rigid, spherical styrene-divinylbenzene copolymer, 5 to 10 in diameter.
A cation exchange resin made of porous polystyrene gel with sulfonic acid groups, about 10 in size.
An ion exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary
ammonium groups, about 10 in size.
A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the
matrix surface, 32 to 63 in diameter.
Packing having the capacity to separate compounds with a molecular weight range from 100 to
5000 daltons (as determined by polyethylene oxide) , applied to neutral, anionic, and cationic
water-soluble polymers. A polymethacrylate resin base,crosslinked with polyhydroxylated ether
(surface contained some residual carboxyl functional groups) was found suitable.
Butyl silane chemically bonded to totally porous silica particles, 5 to 10 in diameter.
Porous silica particles, 30 to 50 in diameter.
A multifunctional support, which consists of a high purity, 100, spherical silica substrate that has
been bonded with anionic(amine) functionality in addition to a conventional reversed phase C8
functionality.
Gamma alumina, reversed phase, low carbon percentage by weight, alumina-based polybutadiene
spherical particles, 5 indiameter with a pore diameter of 80.

Types of columns according to USP (3)

USP Chromatography Column Classifications
L30 Ethyl silane chemically bonded to a totally porous
silica particle, 3 to 10 in diameter.
L31 A strong anion-exchange resin-quaternary amine
bonded on latex particles attached to a core of 8.5
macroporous particles having a pore size of 2000 and
consisting of ethylvinylbenzene cross-linked with 55%
divinyl benzene.
L32 A chiral ligand-exchange packing-L-proline copper
complex covanlently
---L40 Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica
particles, 5 m to 20 m in diameter
----

L51 Amylose tris-3,5-dimethylphenylcarbamate-coated, porous, spherical,

silica particles, 5 to 10 m in diameter
---

10

Outline: How to plan/perform the separation

Fundamental concepts (Basic principles)
Mobile phase parameters
Phase appropriate method development
Troubleshooting
Conclusions

11

Retention
Retention time tR: Time between sample
Injection and analyte reaching the detector
Void time t0: time between sample injection and
unretained substance reaching the detector
void volume x flow rate

tR

t0

h
wb

Peak height h: the height of a peak is

proportional to the injected amount of the
particular component (within linear part of
adsorption isotherm)
Peak width wb: the peak width depends upon
the column efficiency (within linear part of
adsorption isotherm)

Retention times are used for peak identification

12

Capacity factor
Column A: 4.6 x 200 mm

t R t0
k'=
[ ]
t0

5 min
1,5 min

k = 2,33

Retention times are

dependent upon flow rate
and column dimension
Capacity factor k is a
thermodynamic value, thus
independent upon column
dimension and flow rate

Column B: 4.6 x 100 mm

2,5 min
0,75 min

k = 2,33

13

Selectivity

k ' ( 2)
=
k ' (1)

tR (2)
tR (1)
t0

t R (2) t0
=
t R (1) t0

Selectivity is the result of different residence times for

different analytes in the column
Selectivity is dependent upon the nature of the stationary
phase (e.g. C18, C8, phenyl, CN) and the mobile phase
composition

14

Column efficiency, N
The plate model supposes that the chromatographic column
contains a large number of separate layers, called theoretical
plates. Separate equilibrations of the sample between the
stationary and mobile phase occur in these "plates". The
analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.

Physical plate in distillation

columns

Theoretical plate in
rectification columns
(Mc Cabe Tiele diagram)

Theoretical plates in
chromtography columns

15

Column efficiency, N
tR
h
h1/2

w1/2
wb

tR
N = = 16

( )
tR
wb

tR

N = 5.54
w1/ 2

Based on band broadening effects inside the column ( van

Deemter theory), the Gaussian Peak shape broadens with
increasing retention time
The term plate number (count) is a quantitative measure of
the efficiency and is related to the ratio of retention time and
the standard deviation of the peak width.

16

Resolution
The degree of separation
of two neighboring peaks

Rs =

N 1
k '2

1 + k '2

N = 10 000
Rs = 8.33

Rs

tR(1)

tR(2)

N = 2 500
Rs = 4.16

Example:
tR = constant
k1 = 1
k2 = 2

= constant
=2
tR(1)

tR(2)

17

Peak Tailing / Fronting

Different definitions of
peak symmetry are used.

Peak symmetry at 10% peak height

b0.1
As10 =
a 0 .1
As10 > 1
As10 < 1

Tailing
Fronting

Chemical Tailing:

Physical Tailing:

p Mixed interaction mechanisms

p Poorly packed column

p Concentration overload

p Temperature effects

p Poor solubility of the sample in the mobile phase

18

Van Deemter Equation

H, plate heigth:

B
H = A + + C u
u

C
A
u

A: Eddy diffusion

v1
v2
v3
V1 V2 V3

B: longitudinal diffusion
sample diffusion

u: flow rate

C: mass transfer
particle size
pore uniformity

19

Mobile phase parameters

Organic solvent strength and selectivity
Buffers
Acidic mobile phases
Ion-pairing reagents
High pH mobile phases
Other parameters

20

Organic solvent strength

The organic solvent strength and the % controls the retention time
The organic solvent needs to be miscible with water (for RP HPLC)

0.6
THF

elution strength

0.5

ACN

0.4
MeOH

0.3
0.2
0.1
0
0

20

40

60

% organic

80

100

21

Influence of solvent on selectivity

Acetonitrile

Different organic
solvents render different
selectivity

Methanol

p renders narrow, more

symmetrical peaks

p encourages ionic / polar

interactions

p more prone to contaminants

p renders broader, less

symmetrical peaks

p renders less viscous mobile

phases
lower back pressure
p more expensive solvent
p Suitable for neutral
molecules, organic bases
p More hazardous than MeOH

p renders more viscous mobile

phases
higher back pressure
p relatively cheap solvent
p Suitable for stronger acids,
strongly polar metabolites
and isomers

Overall, acetonitrile is the preferred choice of organic solvent in RP-HPLC

22

pH and retention
Examples of ionizable
compounds:
p
p
p
p
p
p
p
p

Amines
Sulfonates
Phosphonates
Carboxylic acids
Sulfates
Phosphates
Thiols
Alcohols

pH

Acidic compounds are

deprotonated
at high pH

Basic compounds are

protonated
at low pH
Ionic compounds are poorly
retained in RP-HPLC

23

pH and retention
Example: Separation of alkaloids
1. lidocaine

pH 2.5
2. papaverine

3. noscapine

pH 10.0
4. diphenhydramine

24

Buffers

Ionization of either basic or acidic APIs greatly influences

the retention
Ionic form generally does not partition into the
hydrophobic stationary phase poor retention
Buffers are used to control the pH of the mobile phase

25

Buffers
Example: Separation of Nor- and Amitriptyline
Nortriptyline

Amitriptyline

Nortriptyline

pH 10
Neutral form is well retained
stable retention
good selectivity
robust method

Amitriptyline

pH 8
Retention time varies strongly within
small pH changes
unstable method

pH 2
No retention, ionic form
does not partition

26

Common HPLC buffers

Buffer

pKA

UV cutoff [nm]

TFA *

0.3

210

phosphate

2.1; 7.2; 12.3

190

citrate

3.1; 4.7; 5.4

225

formate *

3.8

200

acetate *

4.8

205

carbonate *

6.4; 10.3

200

Tris(hydroxymethyl) aminomethan

8.3

210

ammonia *

9.2

200

DEA

10.5

235
* Volatile buffers, MS compatible

27

Important to remember
Buffers are effective within
1 pH unit from their pKA.
Consider the risk of precipitation if high buffer
concentrations are mixed with organic solvents
Always filter buffers through 0.4 m filter prior to use
Be aware of risk for bacterial or fungal growth in pure
aqueous buffers
pH generally shifts upon addition of organic modifier:
p Example: aq. phosphate buffer: pH 7.6
addition of 40% MeOH
pH 8.5

28

Ion-pairing reagents
an alternative to ion-exchange chromatography

Ionic compounds can be

separated by RP-HPLC
N

retention is based on
hydrophobicity of the
ion-pair complex
RP mechanism

N +

broad choice of ion-paring

agents
+N

SiO2

C4

suitable for HPLC mode

29

Influence of flow rate

Pressure drop increases linearly with the flow rate
For isocratic separations, the run time decreases linearly
with the flow rate

H (cm )

The efficiency decreases according to the van Deemter plot

20 m

10 m
5 m
0.00

0.05

0.10
u (cm/s)

uopt

0.15

30

Isocratic vs. gradient elution

Traditionally, most pharmaceutical assays employ isocratic elution

Isocratic elution

Gradient elution

simple technique

more advanced technique

limited peak capacity,

requires analytes of similar
polarity

higher peak capacity,

allows separation of
complex mixtures

prone to injection effects

allows sample
concentration prior to
separation

suitable for small

molecules
parameters such as k and
N are valid

suitable for larger

molecules, e.g. peptides
and proteins
more complex parameters
like k* and P have to be
applied

31

Gradient parameters
Organic solvent range:

starting and final mobile phase composition

Gradient time tG:

time during which the mobile phase composition is changed

Dwell volume:

volume between mixing chamber and column inlet

Peak capacity:

delay

Maximum number of peaks that can fit in a chromatogram with RS = 1

(P tG/wb).
P gradient: 100-200, P isocratic: 50-100

32

Phase appropriate method development

Pre-clinical
phase

Pro-active method
development

Clinical
phase 1 & 2

Level
1&
Level 2
Methods

Secondary
method
development

Clinical phase 3

Level 3
Method
s

Final method
Level 4

Validation

33

Pro-active method development

Objective
p Generation of numerous orthogonal conditions that provide
different separation selectivity

Prerequisite
p Physico-chemical property of API:
structure, solubility / stability, pKA, spectra (NMR, UV, IR, MS),
chirality, potency
p Synthetic route postulation of potential impurities

34

Representative API sample

All significant synthetic impurities and degradation products

should be present.
Subjecting the API to stress conditions according to ICH
guidelines (acidic, neutral, basic pH conditions, light, oxidative
conditions. Parent drug should be degraded by 10-20%

35

Method screening experiments

Goal:
One primary and one secondary method candidate with orthogonal
selectivity by testing different mobile phase systems and different
stationary phases

Orthogonal:
Different elution order due to
e.g. different mobile phase pH

H. T. Rasmussen et. al.; Handbook of Pharmaceutical HPLC Analysis, Separation Science and Technology, vol. 6, Elsevier

36

Method screening experiments

3 - 5 differently surface modified HPLC columns
4 different pH values:
10
10
10
10

mM
mM
mM
mM

NH4Ac / ACN + 0.1% TFA

NH4Ac / ACN + 0.05% HAc
NH4Ac / ACN
(NH4)2CO3 / ACN

premix mobile phases

pH 2.5
pH 4.8
pH 7.0

MS- compatible
mobile phases

pH 9.0

A: buffer / ACN 95/5 (v/v)

B: buffer / ACN 15/85 (v/v)

Run gradient 0 - 100% B / 20 min

37

Selection of candidate method

One candidate method is selected on the basis of
being able to monitor all components of interest
Ideally, no minor peaks elute in the proximity of
the API
A secondary method is also chosen on these
terms, the elution order of the secondary method
has to be significantly different from the primary
method

38

Optimization of candidate method

org. modifier

Test mixture

Column temp.

pH

Gradient time

Buffer conc.

Candidate
method

Optimized method with respect to:

- resolution
- run time
- robustness

Monitor with care:

-

co-elution of impurities
highly retained compounds
unretained compounds
co-elution of impurity and API

39

Additional method parameters

Detection wavelength
- Major components must have suitable dynamic range at selected

wavelength

- All impurities should be detected with suitable sensitivity at selected

wavelength

- Mobile phase should not show strong background absorption

Recommendation
- Use PDA detector during method development
- Modern detectors should have a dynamic range of 4-5 orders of

magnitude

40

Additional method parameters

Sample concentration and injection volume
Such that quantification of major components and impurities at
0.05% level can be performed in a single chromatogram

Sample solvent
Ideally same as initial mobile phase composition, or of weaker character,
otherwise risk for peak deterioration

41

Final method development

Collaboration between development lab (supplier) and QC-lab (customer)

Development lab

QC lab
Planning

Method dev.
Method evaluation
Final method
evaluation

Performance
Feedback

Method transfer
Performance testing

42

Prerequisites for final method development

Synthetic route is locked
All critical intermediates and starting materials are
defined
Formulations and dosage are locked
Relevant impurities and degradation products are known
and available as standards
Certified API standards are available
Specifications for impurities are set, final specifications
are in draft
Representative API batch samples are available

43

Planning
QA

QC

development

regulatory
functions

Planning

MDRD (method definition requirement document):

All stakeholders agree prior to final method development on
the critical attributes of the method

44

Typical criterion for final method

Monitoring API, impurities and degradation products in 1
chromatogram
Run time < 40 min
Precise and robust quantitative analysis
Separation of peaks of interest with RS > 2.0
Detector response for nominal API concentration at 75%
of qualified linear range of detector
Allows easy transfer
Isocratic separation if feasible, otherwise linear gradient
Temperature control room temperature, e.g. 35C

45

Typical criterion for final method

Washing step at the end of each run
Ideally no pH adjustment of buffer
Buffer concentration < 50 mM (risk for precipitation)
Pre-mixed mobile phases
Initial concentration of organic modifier > 5% (v/v)
Globally available HPLC columns
Definition of sample preparation scheme

46

Method development

Review of
available
methods

Comparison
against
MDRD

Development

Optimization /
re-development
of method

Method
evaluation

QC

47

Method evaluation (Pre-validation)

The method is challenged by:
p column durability tests
p batchtobatch reproducibility of columns
p robustness of chromatographic & sample preparation factors
(experimental design with e.g. pH, temperature, gradient, flow
rate, buffer conc. as factors)
p recovery of main component

48

Final method validation

Process of collecting documented
evidence that the method performs
according to the intended purpose1
Classical analytical method validation considers:

accuracy
linearity
specificity
range

ICH Guidelines Q2A

precision
detection
limit

quantification
limit

49

Troubleshooting in HPLC
Poor column performance
Retention time problems
Ghost peaks
Baseline problems
Increased pressure drop

50

Where lies the problem?

Observed problem

Chemistry

Column

Instrument

Stationary phase
Mobile phase
Analyte

Void formation
Uneven packing
Clogged frits
Corrosion

Pump
Injector
Detector
Temperature control
Integration

51

Poor column performance

1. Broad (but symmetrical) peaks
efficiency

Initial or expected performance

poor

observed performance

Either monitor the efficiency of one specific peak of the analysis or perform dedicated
column performance tests periodically

52

Band spreading
Column contribution
van Deemter theory

Extra column contribution

Injection volume
Connection tubing
Detector volume

Dead volumes

Observed bandwidth is the sum of column (col) and extra column (ec) contributions

2 tot = 2 col + 2 ec

53

Injection volume
Small sample amount in large injection volume
volume overload
Recommendations:
Injection volume < 10% of flow rate (mL/min)
Sample solvent should not contain more
organic modifier (RP) than the mobile phase

Peak front remains constant, flat top

and retarded peak tail.

54

Connection Tubing
Separation performance is influenced by the capillary I.D.
by the fourth power !

Using longer capillaries is OK, wider ones will impair the efficiency!

55

Column connection
Make sure the capillaries fit the column!
Fittings from different manufacturers must not be mixed. They differ e.g. in
stop depth.
Pay attention to different threads (British vs. metric system)
Avoid over-tightening
Stop depth

Missmatch

56

Detector flow cell volume

The flow cell volume has a considerable impact on the
plate number
The flow cell is characterized by volume and path length and needs to have the right dimensions
compared to the column:

Column I.D.

Vopt

Vmax

4.6 mm

2.0 L

10 L

4.0 mm

1.5 L

7.5 L

2.0 mm

0.5 L

2.5 L

< 10% peak volume

57

Poor column performance

2. Peak Tailing

Initial or expected performance

Observed performance

58

Identifying the cause of peak tailing

Reduction of retention time
Improved peak symmetry
column overloading

1. Inject less (mass)

2nd component resolves

2 components

Tailing remains

2. Inject neutral compound

(toluene, acetophenone)

Symmetrical peak
secondary interactions
adjust mobile phase pH

Tailing remains
irregularities in column packing

radial temperature gradient

59

Reasons for Tailing

1. Mass overload
entering the non-linear part of langmuirian
adsorption isotherm
cS

cM

Peak appears with tailing

and with shorter retention
time (front side of the peak)

60

Reasons for Tailing

2. Two components that do not resolve
N = 5000

Use smaller particle size

increase N
N = 20000

61

Reasons for Tailing

3. Secondary interactions

few, stronger interactions lead to longer retention times for part of the
analyte population, further more, IEX-interaction often show slow
desorption
tailing

Few, strong ion-exchange interaction,

giving rise to long retention time

+H3N

Many, weaker hydrophobic interaction,

give rise shorter retention times

NH3+

t
NH3+

Peak tailing
t

62

Retention time problems

Poorly reproducible retention times :
- Mobile phase composition
- Temperature
- pH control

Drifting retention time:

-

Equilibration
dewetting of C18 column
stationary phase stability
Irreversible adsorption

63

Mobile phase composition

Clearly state in the method how the mobile phase is to be prepared

V. Meyer, Pitfalls and errors of HPLC in pictures, Hrthig Verlag Heidelberg, 1997

64

Temperature Control
The partition equilibria on which the chromatography is based
are temperature dependent.

Absorbance [Au]

18C

0,8
6.68 min

0,6

9.53 min

7.09 min

0,4

26C

10.23 min

0,2
0
0

10

12

14

Elution time [min]

Kromasil 100-5m-C18 (4.6x250 mm)
MeCN / water 70/30 (v/v)
1 mL/min

1. NaNO2
2. Benzamide
3. Methylbenzoate

4. Toluene
5. Propylbenzene
6. Butylbenzene

65

pH Control
The retention time of acidic and basic compounds depends upon the pH
value.
0.1 pH unit can render a shift in retention time by 10%
Acidic analyte

Basic analyte

35

30

30

25
k' (base)

k' (acid)

25
20
15

20
15

10

10

0
0

pH

+/-

10

12

6
pH

1 pH unit

retention changes by a factor of 5 - 6

10

12

66

Equilibration
Purge column with at least 10 column volumes of mobile phase for at
least 20 min (RP)
For equilibration with buffered mobile phases, allow double time /
volume for equilibration
If you use several mobile phase channels, purge all of them with the
adequate mobile phase
Normal phase columns require ca 10 x longer equilibration time than
RP-phases
Bare silica columns might take hours to equilibrate, pay special
attention to water content in normal phase mobile phases

67

Dewetting
Highly polar mobile phases are expelled from the hydrophobic porous
stationary phase

KR100-5-C18 (4.6 x 250 mm)

100% water
1 mL/min

3,0
2,5
mAU

2,0
1,5
1,0
0,5
0,0
-0,5 0

10

12

14

16

18

20

co-elution of void
marker (NaNO2)
and uracil

reduced void volume

mAU

t / min

3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
-0,5 0

10
t / min

12

14

16

18

20

68

Stationary phase stability & contamination

Inadequate mobile phases can hydrolyze the surface modification
change in surface properties
altered partitioning equilibria

Irreversibly adsorbed compounds block interaction sites

surface area
shorter retention times

reduced

69

Ghost peaks
Peaks that make an unexpected appearance in the chromatogram

Most frequent causes for ghost peaks:

Impurities in the eluent, e.g. excess of ion-pairing agent
Flush the column with HPLC-grade solvents in order to remove the contaminants
Late eluting substances (cross contamination from previous run)
Add a rinsing step with high elution strength after every separation
Memory effect through desorption of a substance from injector seal, fitting or frit
Passivate the system (without column) with conc. NH3, 6M HNO3, DMSO,
acetone, THF
Air bubbles in the detector
Purge with high flow rate (without column), then add a restricting capillary
at the outlet for further use.
Degradation products from an unstable component
Test with another sample, investigate the stability of the original sample
in the mobile phase

70

Baseline problems
Instrumental cause:

- weak or dirty detection lamp

- leaks
- gas in mobile phase or detector cell
- electronic noise
- too high sensitivity

Chemical cause:

- eluting contaminants

Synchronous baseline

Instrumental cause:

- almost always caused by the pump

(air in pump head, valve problems,
broken plunger)

Asynchronous baseline

Instrumental cause:

- mixing problems
- leaks
- gas in mobile phase or detector cell
- electronic noise
- plugged lines

Noisy baseline

71

Baseline problems
Instrumental cause:

- gradient elution
- solvent change
- backpressure changes

Chemical cause:

- contaminated solvents
- compounds eluting from the column

Spikes

Instrumental cause:

- bubbles
- loose wiring
- electrical noise
- malfunctioning lamp relay

Asynchronous baseline

Instrumental cause:

- temperature fluctuations
- mixing problems
- gas in mobile phase
- electrical problems
- erratic pump

Drifting baseline

72

Back pressure problems

Darcys Law, adjusted for non-compressible solvents
P 1 dP 2

u=
L KP
P =

u L KP
dP

P:
L:
:
dP:
KP:

Pressure drop [Pa]

Column length [m]
dynamic viscosity [Pa.s]
mean particle diameter [m]
permeability constant [-]

KP = f() Karman - Cozeny Equation

i
1
1
=

K P (1 i )2 180
3

i:

Interstitial porosity [-]

73

What increases the pressure drop?

x1
Flow
Viscosity
Column length

x2
Mean particle diameter

x3
Interstitial porosity

Pressure drop increases linearly

with flow, viscosity and column length

Pressure drop increases

with 1/d2

Pressure drop
increases with 1/i3

74

In practice that means:

1.

Check porosity!

- Precipitation of compounds decreases the bed porosity

- Also the frit porosity can be decreased (ca 80% of all back pressure
problems are caused by clogged frits.)
Wash the columns with suitable solvents
Only use filtered samples and buffers

2.

Check the particle size!

- Using half of the particle size gives 4 x higher back pressure

3.

Check column dimensions!

- length
- diameter (4.0 vs. 4.6 mm)
column cross section influences the linear
flow rate, thus the back pressure

4.

Check flow rate!

5.

Check viscosity!

- mobile phase composition

- temperature

75

Conclusions
The sample must be soluble if it is not in
solution, it cannot be analyzed by HPLC
Solubility issues complicate real assays of low solubility drugs
and formulations
Many common problems such as lack of mass balance, low recovery and
out of specification results are often based on solubility problems

For separation, analytes must be retained and

have different migration in the column
Suitable choice of mobile and stationary phase for the analysis in question

76

Conclusions, contn
The mobile phase controls the HPLC separation
While the stationary phase provides retention and influences the separation
mechanism, it is the mobile phase that controls the overall separation.
HPLC method development efforts should focus on finding the most adequate
mobile phase

The final analyte should be prepared in the mobile

phase
The analyte should be prepared in the mobile phase or in a weaker analogue
of the mobile phase
Many chromatographic anomalies such as splitted peaks or peak fronting are
caused by injecting the analyte dissolved in a strong solvent

77

Recommended Reading
Handbook of Pharmaceutical Analysis by HPLC; Separation Science
and Technology, Volume 6, edited by S. Ahuja and M. W. Dong;
Elsevier Academic Press; 2005
V. Meyer; Practical High Performance Liquid Chromatography; John
Wiley Sons, 1998
S. Kromidas; More practical problem solving in HPLC; 2005; WileyVCH Verlag GmbH; Weinheim

78

About Kromasil/AkzoNobel
Larger manufacturer of high performance spherical silica
for analytical to industrial HPLC
Fully integrated from raw material to ready packed
columns
Easy traceability when problems
Complete documentation: from manufacture to column
test
Easy traceability when problems
Same properties of particles for analytical to industrial
HPLC
Easy method development
Method development on request
Dedicated, sole distributors in important markets

79

Direct Scalability
from Analytical to Preparative Scale
Particle size distribution
from one Kromasil
silica batch
Kromasil 13 m C18

Kromasil 5 m C18

3.5

10

13

16

Particle size [m]

80

Cornerstones of Quality Assurance

ISO 9001:2000 - Certification
p Validation
p Change Control Policy
p Quality Control

Customer support file

p Audits
p Improvement reports
p Corrective and Preventive actions
p Training

81

Cornerstones of
Safety, Health and Environment
ISO 14001
Responsible care
SHE policy
Near misses reporting
Risk assessments
SHE rounds

82

Customer technical support

Optimization of HPLC separations
p Method development / optimization
p KromaGuide simulations and optimizations

Troubleshooting
p Helping to avoid separation problems
p Solving separation problems

DAC column packing

p Testing of packing methods
p Assisting in column packing

Workshops and seminars at

customer locations

83

Kromasil columns marketed by Tedia in Brazil

RP: C4, C8, C18, Phenyl
NP: SIL, CN, NH2, DIOL, HILIC
Chiral: AmyCoat, CelluCoat, DMB, TBB
SFC: SIL, DIOL, CN, NH2
Composite: Eternity C18 and PhenylHexyl
Columns ID: 2.1mm 50mm(semiprep)
Particle sizes: 2.5 (UHPLC); 3.5, 5 (HPLC);
5,7,10,13, 25 (PREP)

84

The way to peak performance in

liquid chromatography