Professional Documents
Culture Documents
Luqman Hikmattulloh
Product Sales-CSD and Application Manager
Chip LC
Page 2
Nano LC
Capillary
LC
Analytical
LC
Prep LC
Page 3
1. Pressure Issues
Column
Observations
Large
Pressure
Increase
Potential
Problems
Plugged Inlet
Frit
Column
Contamination
Plugged
Packing
Page 4
Back flush column may clear plugged column inlet frit, may not
be possible (i.e. sub 2um columns), check column info
Column Cleaning:
Flush with stronger solvents than your mobile phase.
Make sure detector is taken out of flow path.
Reversed-Phase Solvent Choices
in Order of Increasing Strength
Use at least 10 x Vm of each solvent for analytical columns
1.
2.
3.
4.
Page 6
Page 8
Split Peaks
Can be caused by:
Column contamination
Partially plugged frit
Column void (gap in packing bed)
Injection solvent effects (injecting too much of
a strong solvent
Page 9
Split Peaks
Column Contamination
Column: StableBond SB-C8, 4.6 x 150 mm, 5 m
Mobile Phase: 60% 25 mM Na2HPO4, pH 3.0 : 40% MeOH
Temperature: 35C Detection: UV 254 nm
Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP
Injection 1
Injection 1
After Column Wash
with 100% ACN
Injection 30
4
1
1
4
Time (min)
10
15
Time (min)
10
15
Time (min)
10
15
Column washing eliminates the peak splitting, which resulted from a contaminant on the column.
Page 10
Split Peaks
A. Injection Solvent
100% Acetonitrile
B. Injection Solvent
Mobile Phase
2
2
1
10
Time (min)
Time (min)
10
Injecting in a solvent stronger than the mobile phase can cause peak
shape problems, such as peak splitting or broadening.
Note: earlier peaks (low k) most affected by splitting, later peaks broader
Page 11
Normal
Page 12
Doublet
Peaks
Page 13
Page 14
Normal
Tailing
Normal
Page 15
Tailing
Peak Tailing
pH 3.0
USP TF (5%)
4. 1.33
0.0
2
4
pH 7.0
USP TF (5%)
4. 2.35
5
5
4
2.5
5.0
Time (min)
0.0
2.5
5.0
Time (min)
Reducing the mobile phase pH reduces interactions with silanols that can cause peak
tailing. No additional mobile phase modifiers required.
Consider bonded phase with more endcapping, designed for good pH 7 performance.
Page 16
Peak Tailing
Column Contamination
Column: StableBond SB-C8, 4.6 x 250 mm, 5m
Mobile Phase: 20% H2O : 80% MeOH
Flow Rate: 1.0 mL/min
Temperature: R.T.
Detection: UV 254 nm
Sample: 1. Uracil 2. Phenol 3. 4-Chloronitrobenzene 4. Toluene
QC test forward
direction
Plates
1.
2.
3.
4
7629
12043
13727
13355
TF
2.08
1.64
1.69
1.32
Plates
1.
2.
3.
4
TF
7906
12443
17999
17098
1.
2.
3.
4
1.43
1.21
1.19
1.252
Page 17
2.5
Time (min)
1.06
1.21
1.11
1.17
4
4
0.0
7448
12237
15366
19067
TF
5.0
0.0
2.5
Time (min)
5.0
0.0
2.5
Time (min)
5.0
mAU
60
0.001 mg/mL
0.003 mg/mL
0.005 mg/mL
0.01 mg/mL
0.05 mg/mL
0.1 mg/mL
Pw = 0.2481
40
Pw = 0.2062
20
mAU
Pw = 0.1827
Pw = 0.1840
0
8.2
8.4
8.6
8.8
9.2
9.4
9.6
min
3
2.5
2
1.5
1
0.5
0
8
Pw = 0.1835
8.5
9.5
10 min
Confidentiality Label
pH 4.4
pH 3.0
CH2CH(CH3)2
Ibuprofen
pKa = 4.4
4
5
6
Time (min)
10
4
5
6
Time (min)
10
Inconsistent and tailing peaks may occur when operating close to an analyte pKa and mobile
phase pH should be selected to avoid this.
Page 19
Page 20
Page 21
mAU
40
Problem:
All peaks tail
(top chromatogram).
30
Before troubleshooting
Tailing peaks
20
10
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20... DATA; MERCURY 2010 ECLIPSE PLUS 3X100,1.8\USUYB0145300037.D) mAU
0
2
4
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20...AND PRESS LOAD ECLIPSE PLUS C18 3X100, 1.8\USUYB0145500037.D)
40
500
30
400
10
min
After troubleshooting
Symmetrical Peaks
mAU
600
20
300
10
200
100
10
min
0
2
4
DAD1 A, Sig=254,4 Ref=360,100 (MERCURY 20...AND PRESS LOAD ECLIPSE PLUS C18 3X100, 1.8\USUYB0145500037.D)
10
min
Cause:
Capillary tubing connecting ALS and column was swaged improperly on the ALS
end (ferrule was flush with end of tubing, causing a void).
mAU
600
500
400
Solution:
Replace tubing (bottom chromatogram).
300
200
100
0
2
10
min
Page 22
Peak tailing/fronting
Description
5067-4733
5067-4738
5067-4739
Picture
Fitting Description: Stainless steel screw, an internal stainless steel ferrule, and a front ferrule in PEEK
Where to Use It:
Anywhere in the flow path
Ideal for the connection between the heat exchanger and the column
because it can be re-used without losing tightness.
On competitive instruments
What Does it Replace: The standard stainless steel Swagelock fitting
Why:
Because the heat exchanger had to be replaced when changing the column if the nonremovable Swagelok fitting was used
Page 25
Page 26
1.
2.
3.
4.
Mobile
Phase
Extra Column
Volume
Data
Device
30
25
20
15
N 10500
10
QC Test Conditions:
55% CH3CN
45% H2O
Isocratic, 0.6 mL/min
1 uL injection of QC Mix
23 oC
254 nm
5
0
0.2
mAU
0.4
0.6
0.8
1.2
1.4
min
30
25
20
15
N 4500
10
In 50/50 MeCN/Water
5
0
0.2
0.4
0.6
0.8
1.2
1.4
min
QC test of a 2.1 x 50 mm, 1.8-m Eclipse Plus C18 showing the peak broadening
when larger volume tubing is installed between the autosampler and column.
43% of the efficiency lost with too much extra column volume
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.17
180
280
130
90
105
150
280
400
SS
SS
SS
SS
SS
SS
SS
SS
Green
Green
Green
Green
Green
Green
Green
Green
Connections
1endpreswaged
1endpreswaged
1endpreswaged
preswaged
Withoutfittings
Withoutfittings
Withoutfittings
Withoutfittings
PartNumber Volume(ul)
G131387304
2.0
0109087610
3.2
0109087611
1.2
G131587312
1.7
50211820
1.2
50211821
1.7
50211822
3.2
50211823
4.5
1endpreswaged
1endpreswaged
1endpreswaged
1endpreswaged
Withoutfittings
Withoutfittings
Withoutfittings
Withoutfittings
G131387305
0109087304
0109087305
G131687300
50211816
50211817
50211818
50211819
4.1
6.4
2.9
2.0
2.4
3.4
6.4
9.1
16% increase
60% increase
16% increase
9.6% increase
91% increase
60% increase
A: H2O; B: CH3CN
0.4 mL/min
t (min)
0
1.2
%B
95
25
30000
25000
20000
N
15000
10000
5000
0
0
10
15
20
25
30
Volume of Tubing between ALS and Column (L)
35
40
The above scatter plots compare the effects of column length and internal diameter on
extra column volume; length has only a slight effect, while internal diameter is greatly
affected by extra-column volume.
UV
Data Collection Rate
0.12 s
mAU
300
100
0.25
0.25 s
mAU
0.5
0.75
1.5
1.75
50 ms
1
100
0.25
0.5
0.75
1.25
1.5
1.75
min
100 ms
1
PW1/2=0.019
200
PW1/2=0.024
S/N=43
x106
300
1.0 s
0.25
0.5
0.75
mAU
300
1.25
1.5
1.75
min
x106
250 ms
1
PW1/2=0.025
200
PW1/2=0.035
S/N=118
100
0
0
2.0 s
mAU
0.25
0.5
0.75
1.25
1.5
1.75
min
200
x106
500 ms
1
PW1/2=0.042
300
0.25
0.5
0.75
1.25
1.5
1.75
min
PW1/2=0.049
S/N=109
100
0
0
PW1/2=0.027
S/N=86
100
0
0
PW1/2=0.024
S/N=39
min
PW1/2=0.018
200
0.5 s
mAU
1.25
25 ms
x106
300
0
0
x106
PW1/2=0.018
200
0
0
MS Scan Rate
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
Flow Cell
Flow Cells are an integral part of HPLC instrumentation.
Choose the best one for the column used
Dont just use the largest one available
Peak broadening will compromise sensitivity and detection limits
20000
Efficiency (N)
10000
k=3.8
5000
0
0
4
6
8
10
Flow Cell Path Length (mm)
12
1 ul QC Mix, Uracil, Phenol (k=0.5), 4-Chloronitrobenzene(k=2), Napthalene (k=3.8) 55% MeCN 45 % Water 0.55 ml/min micro flow cell
Reproducibility
Use the column oven to improve precision
Typically,
Area and Peak Height problems together point to the autosampler system
Area and Retention Time problems together point to the pump
Page 37
3. Retention Issues
Page 38
Column aging
Column contamination
Insufficient column equilibration
Poor column/mobile phase combination
Change in mobile phase
Change in flow rate
Change in column temperature
Other instrument issues
Change in Retention/Selectivity
Column-to-Column
1. Different column histories (aging)
2. Insufficient/inconsistent equilibration
3. Poor column/mobile phase combination
4. Change in mobile phase
5. Change in flow rate
6. Other instrument issues
7. Slight changes in column bed volume (tr only)
Page 40
Evaluate:
1. All causes of column-to-column change*
2. Method ruggedness (buffers/ionic strength)
3. pH sensitivity (sample/column interactions)
*All causes of column-to-column change should be considered first,
especially when only one column from a lot has been tested.
Page 41
Confidentiality Label
42
Problem:
The selectivity was different on each of the columns
Confidentiality Label
43
15
10
1.635
5um
5,6 = 2.70
3.907
20
1.229
0.293
0.360
25
0.712
mAU
0.961
N=3800
5
0
-5
20
15
5
0
min
min
4.198
0.017
10
3.5um
5,6 = 2.78
1.290
0.300
0.369
25
1.722
mAU
0.996
1
0.725
N=7100
-5
15
10
5
4.897
1.8um
5,6 = 2.88
1.389
0.299
20
0.784
mAU
25
2
1.900
1
1.071
N=9900
0
-5
0
44
min
1.612
1.615
0.695
0.696
3.873
3.879
0.944
0.946
mAU
46 bar
0.352
0.353
15
1.213
1.214
0.2880.288
20
10
= 2.71
0.249
-5
1
1.668
1.666
0.970
0.971
mAU
0.713
0.714
30
0.290
4.073
4.072
25
15
= 2.74
1.241
1.241
0.287
20
246 bar
0.348
0.351
0.848
0.417
10
-5
45
min
5um
2.70
2.71
3.5um
2.75
2.74
1.8um
2.88
2.74
Page 46
Conclusions:
Most HPLC column problems are evident as:
1. High pressure
2. Undesirable peak shape
3. Changes in retention/selectivity
To maximize column lifetime and minimize downtime:
1. Prevent pressure problems with in-line filters and
appropriate sample prep.
2. Use appropriate fittings and make good connections,
optimize the U/HPLC for maximum efficiency
3. Use appropriate
Page 47
Anarcoticeffect,causingfatigue,dizziness,nausea,vomiting,worseningof
asthmasymptoms
Irritationoftheeyesandtherespiratorytract
Dermatitisandotherskindisorders
Damagetotheliver,kidneys,heart,bloodvessels,bonemarrowandthe
nervoussystem,cancer
Unlessyouarewearingthis
Ifnot,wehave
SolventSafetyCaps
TestConditions
BottleAThisBottlewasclosedwithaSafetyCap,fittingpreciselythe
standardGL45glassbottlethreadused
BottleB ThisbottlewastightlyclosedwiththeGL45capprovided
includingaTeflonfoilseal.
BottleC Thisbottlewasclosedwithacaphavinga10mmhole
leavinganopeningofanareaofapproximately0.785cm2.
BottleD Thisbottlewasclosedwithacaphaving3holes,3mmeach,
leavinganopeningofanareaofapproximately0.212cm2.
Procedure
Atthestartofthetest,all4bottleswerefilledwiththesame
mixtureofWater+Methanol=20+80(w/w).
UsingBottleBasareference,chromatogramsofamixtureofthe
threePAHs(Naphthalene,Pyrene,Chrysene),werecomparedwith
thereference.
Afterthefirstmeasurement,allbottleswerekeptatroom
temperaturewithagentleairflowfor31days.
Results
HPLCresults
SolventSafetyCaps
ForNS29/32solventbottles
ForGL45solventbottles
Forwastebottles
TheAgilentsafetycapsarefreely
rotating,PTFEandPFAscrewcapsfor
allsolventbottlesandwaste
containersusedwithHPLCsystemsin
thelab.
HowtoconnectaSafetyCaponaBottlewithadifferent
thread
SolventSafetyCapsforwastebottle
Changefilterafter6months!
TipsonHPLCMobilephases(shouldyoudonothavethe
safetycaps)
Anymobilephasethathasanaqueousphaseinitshould
bediscardedwithin1dayofuseunlessaspecialadditive
isincorporatedtopreventmicrobialgrowth.
MobilephasesthathasbeenpHadjustedshouldonlybe
usedfor1day.
pHshouldbecheckedandnewadjustmentsmustbe
madeifthereispHconcerned.
Page 61