!!"#

$ $

%

$&

$ (

$!)*+

$", $

-"",$! -"",$!,

"!,"$!

.

!

Column L Particle size Flow rate Rel. time Rel. R s Rel. P

250 mm 10 m 0.5 ml/min 1 1 1

125 mm 5 m 1.0 ml/min 0.25 1 4

87.5 mm 3.5 m 1.43 ml/min 0.125 1 8

62.5 mm 2.5 m 2.0 ml/min 0.0625 1 16

45 mm 1.8 m 2.8 ml/min 0.0321 1 32

/

)0+

*"*)+ 123425

.606

77)87+ --9 -79:79--

)9-+ )9-+ $

9

(()77! 8 --9:79

1

)+

-;%-;?>>

4

!" #$%&

069$ /02@8

069$ $$2/2@ 8

/02@8 ( $ 2/2@8 ) $ 2/2@8 3%#3 $

2/2@82/2@8 69$ /02@8 * 7 $

02@8 +

6

!" #$%&

$ /02@8 3% 3#3"$

00@8 * $ /02@8 + 3% 3#3"$

B@8 %$ /02@8 3"$ /02@8 % !$02@8 % ! % !

$02@8 ( 3" !%

%1@8 ) #"! !022

/222)$%+ !3$8$ #!%)$% +!$8

C$ /02@8 2/2@8 * ! 022D$

$$!)+ ""68

+ E"!$ $!3$$/@! 62D8

B

!" #$%&

;$02@8

3%3$%68/ @"222D "$3#!/ /5"$8"$8

3%#33%"

3333 ( 3/3$

/A2A 3333

) 3/3$3 /02A

333

02

()C+

$

"

$

9F!,

$

00

*

t0

tR

h

Retention time t R: Time between sampleInjection and analyte reaching the detector

Void time t 0: time between sample injection andunretained substance reaching the detector void volume x flow rate

Peak height h : the height of a peak is proportional to the injected amount of the

twb

proportional to the injected amount of theparticular component (within linear part ofadsorption isotherm)

Peak width w b: the peak width depends upon the column efficiency (within linear part ofadsorption isotherm)

Retention times are used for peak identification

0

k = 2,331,5 min

5 min

t

Column A: 4.6 x 200 mm

][0

0 --

=t

ttk R

*!

#G"!

t

Column B: 4.6 x 100 mm

0,75 min

2,5 min

t

k = 2,33

0

"H

t0

tR (1)

tR (2)

)1()2(

kk

=a

0

)1()2(

ttttR

--

=a

"

" )880667+$

t 0)1( ttR -=a

0.

7

$ 8$$! $II 8"!$ $$%8

) $+

0/

7

( )22

16 RwtRtN =

=s

t

h1/2

h

w1/2

tR

wb

2

54.5

=t

N R

C$$ ) "+E#$!

$)+" "#!8

( )16bw

N =

=s 2/1

54.5

=w

N

01

*

!$# N = 10 000

Rs = 8.33

tR(2)ttR(1)

sR2

11

4 kkN

Rs +

-=

aa

N = 2 500Rs = 4.16

tR(2)t

tR(1)

tR(2)tR(1)

214 kRs +

=a

,-

* J#G0 J0#G J

J J

04

#,(

!

.""""/ 0

1.0

1.010 a

bAs =

/1/2#2

K>F %

"

$$

1.0a

K>F #

06

L;

C

uCuB

AH ++=

F

F ,""

V1 V2 V3

CF# "" M

F " particle size pore uniformity

v1

v2

v3

0B

$

9""

C

$

-3

$

9

2

"5

"$$!! )*+

28/

281THF

ACN

9"

2

280

28

28

28.

28/

2 2 .2 12 62 022

% organic

elut

ion

stre

ngth

ACN

MeOH

0

-""

32

"

"

!#

"$

,

$#

"$

452"2" #56

!$#

%""

$$

9

"$ $#

""

$$

;%$F

$%

2#2

7 6

8

0

0 5

;%F#

0)

0

(0 22

0

.

C

-$-

-$ $

C$

/

C;%F73

7! $ " !

7

7

7

**"! !

1

"" 9 $: "";

!2

#

"

#!%%

Q

$

#G7"

%%

!

$88

%##N"$

0

9"F$

E EF!$

!"F"$!%$

E

"$!%$

#F %$# !* J0)R E,!$+8 F022322 F/23022

6525

5?

5

32

5

32

@25(

62

22?

"

32

2

5

:

3""

9$Q" E "

"

3-F

$,$& )7 *?L-* +

.

*"-

$8

$Q- -)$ %"8$$02 3258$$02 325

/

EF9 !"$$

42#

%

"##$##%"! & "(!( )#*(+)

42#

."" 0#0""A2

1

3 /

."F

02 7 . ,7S2805( 8/02 7 . ,7S282/5 .86 3 6 A

%

02 7 . ,7S282/5 .8602 7 . ,7 48202 )7 . + 9 ,7 B82

%$ F$,7B/,/)","+

CF$,70/,6/)","+

*23 0225C,2

3 6 AA2

4

9$$$

-#%-

$

6

2

>

#0

"

0

""

0

!

-

382

3 6 " 3 2#2 3 3 6 " 7

9

>

#0

"

0

""

0

!

-

42826 6 6 A

3 6 " 7

B

!"3 Q"$

!"

3 $!$ "!"

3 $!$#$

3 $!$#$

*3 ?"

3 ".3/

.2

Q"Q 282/5"$

"-$ !#!##

.0

$$!"$)+ T3$)+

Development lab QC lab

Planning

Method dev.

("

Final methodevaluation

Method evaluation

Method dev.

PerformanceFeedback

Method transfer

Performance testing

.

"

#

(#

*"# ! *"# !"$

-"$

*"-$"$

.

QA regulatoryfunctions

developmentQC

Planning

MDRD (method d efinition r equirement d ocument):

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

..

- 0

*U.2

$"

#!*V82

#!*V82

- 4/5

!

-$!

W88/X

./

=

-Q$

CU/2 )# +

3%$

-V/5)","+

E$"$

.1

58"5A2

#3..

4=65"2

325

"

.5 QC

.4

") 3"+

$F

$

$YY$$

$Z )%!88 !)%!88 !$8+

"

.6

Process of collecting documentedevidence that the method performsaccording to the intended purpose1

Classical analytical method validation considers:

("

"

B "#

1 ICH Guidelines Q2A

.B

*$

E#

C$

$

-

/2

Observed problem

=$[

Chemistry

Stationary phase Mobile phase Analyte

Column

Void formation Uneven packing Clogged frits Corrosion

Instrument

Pump Injector Detector Temperature control Integration

/0

08C)$+#

Initial or expected performance observed performance

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

/

$ 5.2

;%$ 7C5 A# Dead volumes

C

A# .5

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

ec2

col2

tot2 s+s=s

Dead volumes

/

Q" ""

Recommendations:

-Q"

Peak front remains constant, flat topand retarded peak tail.

Injection volume < 10% of flow rate (mL /min)

Sample solvent should not contain moreorganic modifier (RP) than the mobile phase

/.

$ -88$!\

$

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

//

#\

Fittings from different manufacturers must not be m ixed. They differ e.g. in stop depth.

Pay attention to different threads (British vs. met ric system)

Avoid over-tightening

Missmatch

Stop depth

/1

!"$ $

The flow cell is characterized by volume and path l ength and needs to have the right dimensionscompared to the column:

!"

-88 L L %

(0 0D D

(0 0)D 0)D

0 0)D 0)D

< 10% peak volume

/4

8#

Initial or expected performance Observed performance

/6

1. Inject less (mass)

Reduction of retention time Improved peak symmetry

column overloading

2nd component resolves 2 components

Tailing remains

-#

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

/B

08 "#26 "# 2

cS

*

cM

Peak appears with tailingand with shorter retentiontime (front side of the peak)

12

8!"

N = 5000

Use smaller particle size

*

N = 20000

Use smaller particle size increase N

10

8! -;

1

$F3 $3 3

F

*$

F3 ;$3 !063 $3 -"$

1

!$ $

$

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

1.

$! $8

0,4

0,6

0,8

1

Abs

orba

nce

[Au]

18C 26C

10.23 min

9.53 min6.68 min

7.09 min

0

0,2

0,4

0 2 4 6 8 10 12 14

Elution time [min]

Abs

orba

nce

[Au]

10.23 min7.09 min

Kromasil 100-5m-C18 (4.6x250 mm) 1. NaNO 2 4. TolueneMeCN / water 70/30 (v/v) 2. Benzamide 5. Propylbenzene1 mL/min 3. Methylbenzoate 6. Butylbenzene

1/

$ "8

280$ 025

25

30

35

25

30

Acidic analyte Basic analyte

0

5

10

15

20

25

0 2 4 6 8 10 12

pH

k (

aci

d)

0

5

10

15

20

0 2 4 6 8 10 12

pH

k (b

ase)

+/- 1 pH unit retention changes by a factor of 5 - 6

11

!02"$ 2)*+

($!$$ !$,"$

-"$ !$

;$

702%$ *3

C#$ !$

14

$% $

0,5

1,0

1,5

2,0

2,5

3,0

mA

U

KR100-5-C18 (4.6 x 250 mm) 100% water 1 mL/min

!

-0,5

0,0

0 2 4 6 8 10 12 14 16 18 20

t / min

-0,50,00,51,01,52,02,53,03,5

0 2 4 6 8 10 12 14 16 18 20

t / min

mA

U co-elution of voidmarker (NaNO 2) and uracil

reduced void volume

16

-$ $

-"$$$#

$Z

-"$$$#

1B

Peaks that make an unexpected appearance in the chr omatogram

Most frequent causes for ghost peaks:

Impurities in the eluent, e.g. excess of ion-pairin g agent Flush the column with HPLC-grade solvents in order to remove the contaminants

Late eluting substances (cross contamination from p revious run) Add a rinsing step with high elution strength after every separation

Memory effect through desorption of a substance fro m injector seal, fitting or frit

E#

Memory effect through desorption of a substance fro m injector seal, fitting or frit Passivate the system (without column) with conc. NH 3, 6M HNO3, DMSO,

acetone, THF

Air bubbles in the detector Purge with high flow rate (without column), then ad d 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

42

Instrumental cause: - weak or dirty detection lamp- leaks- gas in mobile phase or detector cell- electronic noise- too high sensitivity

Chemical cause: - eluting contaminants

Noisy baseline

C$

Synchronous baseline Instrumental cause: - almost always caused by the pu mp(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

40

Instrumental cause: - gradient elution- solvent change- backpressure changes

Chemical cause: - contaminated solvents- compounds eluting from the column

Drifting baseline

C$

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

4

Darcys Law, adjusted for non-compressible solvents

2

21

P

P

P

P

d

KLuP

Kd

LP

u

=D

D

=

hhhh

hhhh P: Pressure drop [Pa]L: Column length [m] : dynamic viscosity [Pa.s]dP: mean particle diameter [m]KP: permeability constant [-]

C#$

Pd

KP = f( ) fi Karman - Cozeny Equation

( ) 1801

1

12

3

-

=i

i

PK eeee

eeee i: Interstitial porosity [-]

4

FlowViscosity

Column length

x1

x2

Pressure drop increases linearlywith flow, viscosity and column length

=[

Mean particle diameter

Interstitial porosity

x2

x3

Pressure drop increaseswith 1/d 2

Pressure dropincreases with 1/ i3

4.

08 #\3 $ 3 $)625 $#

$$8+ =!$" 9$

8 #\3 ?".%$ #

-F

3 ?".%$ #

8 #\3 3 ).82"8.81+

!$#

.8 #!\

/8 #" \3 $3

4/

$$Y $$

Solubility issues complicate real assays of low sol ubility drugs and formulations

Many common problems such as lack of mass balance, low recovery and

Many common problems such as lack of mass balance, low recovery andout of specification results are often based on sol ubility problems

($"

Suitable choice of mobile and stationary phase for the analysis in question

41

$

While the stationary phase provides retention and i nfluences the separationmechanism, it is the mobile phase that controls the overall separation.

HPLC method development efforts should focus on fin ding the most adequatemobile phase

G

$$

The analyte should be prepared in the mobile phase or in a weaker analogueof the mobile phase

Many chromatographic anomalies such as splitted pea ks or peak fronting are caused by injecting the analyte dissolved in a stro ng solvent

44

$#$O L1$8Q 8 =8O;"O22/

L8 O O]=0BB6

**

8&O $" O22/O=3LLE$O=

46

$&,#7$

(!# ;$!$

F ;$!$ ;$!$

;"

"

$#

4B

$"

$&$

;D

62

T

-9B220F2223 L

T

-"

"""

60

;"

-90.220

*$

;

7

*# *#

;

6

9 ", &E

$ "$ "$

# # #

=#

6

$C

*F.606

7F-77-9--

F CCC

(F--977

F;06% F;06%

-F80Y /2)+

F8/A)?+O8/A/A)+O/4020/)*;+

6.

!#!#