presentation analytical hplc
DESCRIPTION
Química Analítica - HPLCTRANSCRIPT
-
!!"#
$ $
%
$&
-
$ (
$!)*+
$", $
-"",$! -"",$!,
"!,"$!
-
.
!
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.
!#!#