Advanced
Ad
d Topic:
T i Tips
Ti
and Tricks of LC User
Maintenance
Edward Kim
Application Engineer
February 19
19, 2010
Agilent 1200 Series LC – Agilent Restricted
Page 1
2/18/2010
mA
U
Troubleshooting in HPLC
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Page 2
0
5
1
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Time
(min)
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Agilent 1100/1200 Series System
Operating Principles
Pumping principle
Sampler principles
TCC principle
Detector principle
Agilent 1200 Series LC – Agilent Restricted
Page 3
2/18/2010
Fast Serviceability – Still Unmatched
Savings from E-PAC Design:
f60% reduction in mechanical
parts
f90% reduction in screw joints
f70% reduction in assembly time
f90% reduction in disassembly
time
E-PAC: Expanded Polypropylene foam
All pure materials for low cost recycling
Agilent 1200 Series LC – Agilent Restricted
Page 4
2/18/2010
Agilent 1100/1200 Module
Serial Number Format
10 characters:
02274
DE
916
Where made?
Date code
•JP = Japan
•DE = Germany
•US = USA
•CN = China
•FR = France
IIndividual
di id l module
d l counter
t
(how many have been made?)
(16st week of 1999)
• Each module has it's own module counter.
• The date code marks a major change in production
production.
• The module counter starts at about 100 for each type of module
and continues in order regardless of changes to the date code.
• Individual module counter higher 55000 refers to 1200 Series
lower 55000 to 1100Series.
Agilent 1200 Series LC – Agilent Restricted
Page 5
2/18/2010
Degassers - Overview
G1322A Vacuum Degasser (aka Standard Degasser/Online Degasser)
G1379A Micro Vacuum Degasser 1100 Series (obsolete)
G1379B Micro Vacuum Degasser 1200 Series
Agilent 1200 Series LC – Agilent Restricted
Page 6
2/18/2010
Standard degasser operating principle
Agilent 1200 Series LC – Agilent Restricted
Page 7
2/18/2010
Avoid growth of algae
Agilent 1200 Series LC – Agilent Restricted
Page 8
2/18/2010
Comparison of degassers
G1322A:
• better for applications using methanol
• recommended for use with refractive index detector (RID)
• better for high flow rates (>5 ml/min)
G1379A/B
• better in all other cases
Agilent 1200 Series LC – Agilent Restricted
Page 9
2/18/2010
Maintenance
• Cleaning – Flush the solvent tubes with
isopropanol.
• When using buffers, flush with water.
• May
M h
have tto exchange
h
th
the vacuum pump,
sensor, solenoid valve, or chamber.
• Replace solvent inlet frits
Solvent
B
A
C
D
Pump
10
Pumps - Overview
G1310A IIsocratic
ti P
Pump: one solvent
l
t ((might
i ht b
be mixture)
i t )
G1311A Quaternary Pump: four solvents, low pressure mixing
G1312A Binary Pump: two (four) solvents, high pressure mixing
[G1312B: binary pump SL (RRLC)
Special pumps for other purposes: cap, nano, prep systems]
Agilent 1200 Series LC – Agilent Restricted
Page 11
2/18/2010
Isocratic pump
Agilent 1200 Series LC – Agilent Restricted
Page 12
2/18/2010
Quaternary/binary pump
Quaternary Pump
Binary Pump
high pressure
mixing
multi-channel
gradient valve (MCGV)
l
low
pressure mixing
i i
Agilent 1200 Series LC – Agilent Restricted
Page 13
2/18/2010
Pump options
• Solvent Selection Valve (P/N G1312-60000)
G1312 60000)
• Normal Phase Seals
(P/N 0905-1420)
• Seal Wash
(P/N 01018
01018-68722)
68722)
• Active Seal Wash
(P/N G1311-68711)
Agilent 1200 Series LC – Agilent Restricted
Page 14
2/18/2010
Seal wash option
Seal Wash, active/passive
Agilent 1200 Series LC – Agilent Restricted
Page 15
2/18/2010
System Pressure Test
Pressure tightness up to 400 bar of the system (this is no pump test!)
Ti ht system
Tighten
t
with
ith blank
bl k nut,
t preferably
f bl att TCC
Use isopropanole as solvent
Pressure increased to 390 bar, decrease lower than 2 bar/minute
Agilent 1200 Series LC – Agilent Restricted
Page 16
2/18/2010
Leak test for quaternary pump
Pressure tightness of the quaternary pump
Tighten the pump outlet with a blank nut
Use isopropanole as solvent, flush system
y
prior to test (remove bubbles)
Steps:
Ramp 1: both plungers deliver for two strokes (1-2-1-2)
Plateau 1: plunger 2 with low flow (2 µl/min)
Ramp 2: Plungers 2 and 1, high flow (153 µl/min)
Plateau 2: Plunger 1 delivers with low flow (2 µl/min)
Ramp 3, 4: Plungers 2-1-2-1 at high flow, up to 390 bar
tightness of pump? degassing efficiency?
2
1
problems at plateau 1
⇒ check OBV
OBV, plunger/seal 2
2,
purge valve, blanking nut
problems at plateau 2
⇒ check AIV, plunger/seal 1
⇒ also affected by chamber 2
Agilent 1200 Series LC – Agilent Restricted
Page 17
2/18/2010
G1312A Leak Test
IF A2 & B2 failed:
everything after the both OBV could be
leaking
g including
g blank nut and p
purge
g valve
Agilent 1200 Series LC – Agilent Restricted
Page 18
2/18/2010
Pump parameters
flow: volume delivered per time
time, range depends on pump type
composition: percentage for each solvent (bin./quat. pump)
stroke:
t k the
th volume
l
iin a range off 20 µll tto 100 µll off compressed
d
solvent delivered by one movement of piston 1
p
mixing
g but decreases lifetime of seals
a lower stroke improves
and valves
stroke
100 µl
auto
settable stroke
auto: minimum possible
stroke for a chosen flow
((recommended setting)
g)
20 µl
0.1 ml/min
5 ml/min
flow
Agilent 1200 Series LC – Agilent Restricted
Page 19
2/18/2010
Pump parameters: compressibility
Compressibility: relative volume change per pressure change
κ = ∆V/(V·∆p), e.g. water 46 ·10-6 bar, acetonitrile 115 ·10-6 bar,
isopropanole 100 ·10-6 bar, see manual/on-line help
is corrected by initial pump speed for avoiding pressure
fluctuations (pressure ripple should be below 1%)
Agilent 1200 Series LC – Agilent Restricted
Page 20
2/18/2010
Using the pressure plot
piston 2 delivers
pressure (ripple)
piston 1 delivers
piston 2 delivers
pressure drop
p
p
Agilent 1200 Series LC – Agilent Restricted
Page 21
2/18/2010
Optimization of compressibility settings
compressibility
ibilit setting
tti ttoo hi
high
h
compressibility setting too low
compressibility changes during gradients = changing composition
prior to mixing for quaternary pumps
Agilent 1200 Series LC – Agilent Restricted
Page 22
2/18/2010
Terms for volumes in HPLC
Delay volume: volume from the first point of mixing to the head
of the column =>
> delay increases time needed for gradient to
arrive at column => elution is delayed
Dispersion volume: volume between the point of injection and
end of flow cell. Mixing dispersion causes peak broadening
The volume between point of injection and head of the column
contributes to both the delay and dispersion volume
volume, ii.e.
e peaks
come later and are broader
=> use short connections and small diameters (columns,
(
,
capillaries)
Agilent 1200 Series LC – Agilent Restricted
Page 23
2/18/2010
Delay and dispersion volumes
for binary and quaternary pump
Quaternary pump: 800 µl to 1100 µl
Binary pump:
180 µl to 480 µl
Largest contribution by damper (1µl/bar)
Agilent 1200 Series LC – Agilent Restricted
Page 24
2/18/2010
Autosamplers - overview
G1313A Autosampler (obsolete)
G1329A Thermostated Standard Autosampler (1100/1200)
[G1329B: ALS SL (RRLC) – high pressure variant]
G1367A Well-Plate
Well Plate Autosampler (1100 Series)
G1367B High-Performance Autosampler (1200 Series),HiP ALS
[1367C: HiP ALS SL (RRLC)]
[1367D HiP ALS SL+ (RRLC,
[1367D:
(RRLC llow carry over als)]
l )]
G1330A/B Autosampler Thermostat
[special samplers for other applications: micro autosampler
autosampler, micro wellplate sampler
sampler, dual-loop
autosampler, prep autosampler CTC]
Agilent 1200 Series LC – Agilent Restricted
Page 25
2/18/2010
Standard ALS Principle
Mainpass
Pump
to Column
Draw Sample
(Bypass)
to Column
Inject
Pump
Pump
to Column
Agilent 1200 Series LC – Agilent Restricted
Page 26
2/18/2010
HiP ALS Principle
Agilent 1200 Series LC – Agilent Restricted
Page 27
2/18/2010
ALS (Std/HiP) Options
• Extended Volume Metering Device 900µl
• MultiDraw Kit 400µl & 1400µl & (5000µl prep)
• Red Needle Seat Capillary 0,12mm ID
Agilent 1200 Series LC – Agilent Restricted
Page 28
2/18/2010
Problems with Reproducibility – Peak Areas
Peak Areas not
Reproducible
With peak height
•Rotor seal cross-port
cross port leak or injection valve not tight
•Piston seal of metering unit leaking
•Needle partially blocked
With retention time
•Variable pump flow rate
Other
•Capillary
p
y from injector
j
to detector not tight
g
•Detector equilibration problems
Page 29
Seal
Pump
Column
Rotor Seal
Thermostatted Column Compartment
- overview
Th
Thermostatted
t tt d Column
C l
C
Compartment
t
t G1316A TCC
[G1316B: TCC SL (RRLC)]
[G1316C: TCC SL+ (Method Devellopment Solution)]
Agilent 1200 Series LC – Agilent Restricted
Page 30
2/18/2010
TCC principle
G1316A
Agilent 1200 Series LC – Agilent Restricted
Page 31
2/18/2010
Agilent 1100/1200 Series Thermostatted
Column Compartment
G1316A
Options:
Column Switching Valve 6 or 10 Port
Specs:
Specs
10 degrees below ambient to 80 deg C
up to 80 deg C: max flow rate 5ml/min
Agilent 1200 Series LC – Agilent Restricted
Page 32
2/18/2010
Agilent Capillary Color Code
C l
Color
id
i.d.
Red
0.12 mm
Green
0.17 mm
Blue
0.25 mm
Note: All fittings in the 1100
HPLC are Swagelok compatible,
EXCEPT the ports on model
77251 Rheodyne valve.
33
Fittings
g
Swagelok
Waters
0 130 iin.
0.130
0 090 iin.
0.090
Rheodyne
Parker
0.170 in.
0.090 in.
Valco
Uptight
0.080 in.
0.090 in.
Troubleshooting LC Fittings Part II,
II
J.W. Dolan and P. Upchurch, LC/GC Magazine 6:788 (1988)
34
Detectors
G1314A Variable Wavelength Detector (1100 Series) VWD obs.
G1314B Variable Wavelength Detector (1200 Series) VWD
[G1314C Variable Wavelength Detector SL (1200 Series) VWD SL]
[G1314D/E Variable Wavelength Detector SL+ (1200 Series) VWD SL+]
G1315B Diode-Array Detector (1100/1200 Series) DAD
[G1315C Diode-Array Detector SL (1200 Series) DAD SL
G1315D Diode
Diode-Array
Array Detector (1100/1200 Series) DAD SL]
G1365B Multiple-Wavelength Detector (1100/1200) MWD
[[G1365C/D Multiple-Wavelength
p
g Detector SL ((1200 Series)) MWD SL
G1321A Fluorescence Detector (1100/1200 Series) FLD
G1362A Refractive Index Detector (1100/1200 Series) RID
G4218A Evaporative Light Scattering Detector (1200 Series) ELSD]
Agilent 1200 Series LC – Agilent Restricted
Page 35
2/18/2010
Lamp Matrix
Detector
New
Original
DAD
G1315A/B/C
MWD
G1365A/B/C
DAD SL
G1315D/E
MWD SL
G1365D/E
1290 Infinity
G4212A (New)
VWD
Secondary
VIS Lamp
2140-0813
5181-1530
(2140-0820)
G1103-60001
2140-0820
5181-1530
(2140-0813)
G1103-60001
5190-0917
NA
G1103-60001
G1314A/B/C
G1314-60100
2140-0813
G1103-60001
VWD SL+
G1314D/E
G1314-60101
2140-0820
G1103-60001
UV VIS
8453A
2140-0605
2140-0813
G1103-60001
CE
G1600A
2140-0585
NA
NA
CE
G7100A (New)
5190-0917
NA
NA
New SL and SL+ detectors shipped with RFID lamp
SL detectors can use lamps without RFID chip (Backwards compatible)
When using a secondary lamp in a VWD, the setting in the detector needs to be changed
The UV-VIS system can run with a long life lamp. The CE system can not.
Lamps with RFID chip can be used with old detectors, but no functionality of the chip.
Page 36
Group/Presentation Title
Agilent Restricted
Month ##, 200X
VWD principle
I0
I
I0
Agilent 1200 Series LC – Agilent Restricted
Page 37
2/18/2010
VWD flow cells
Typical Column
Length
Typical Peak
Width
<= 5 cm
0.025
10 cm
0.05
20 cm
0.1
>= 40 cm
0.2
Recommended Flow Cell
Micro Flow Cell
Semimicro Flow
Cell
High-Pressure
Flow Cell
Standard Flow
Cell
For Pressure
Above 100 bar
Typical Flow Rate
0.05-0.2 mL/min
0.2-0.4 mL/min
0.4-0.8 mL/min
1-2 mL/min
Internal Column Diameter
1.0 mm
2.1 mm
3.0 mm
4.6 mm
0.05-5 mL/min
Agilent 1200 Series LC – Agilent Restricted
Page 38
2/18/2010
Baseline stabilization
VWD baseline shows a slight
g drift for the first 60 minutes:
Agilent 1200 Series LC – Agilent Restricted
Page 39
2/18/2010
VWD diagnostic tests
Agilent 1200 Series LC – Agilent Restricted
Page 40
2/18/2010
Wavelength calibration
Or: how to calibrate the rotation angle of the grating
0
1
1
656 nm
=>
>T
Two positions
iti
needed:
d d zero order
d and
d fifirstt order
d 656 nm emission
i i liline
of deuterium lamp
required after repair, part replacements or for PM
the 656 nm position is checked each time the lamp is switched on
Agilent 1200 Series LC – Agilent Restricted
Page 41
2/18/2010
VWD: Holmium oxide test
Similar to wavelength calibration, done for 3 absorption lines of holmium filter
Verifies success of wavelength calibration, used in OQ/PV and
after flow cell maintenance/repair
Agilent 1200 Series LC – Agilent Restricted
Page 42
2/18/2010
VWD intensity test
Measures intensity for complete wavelength
range and for a flow cell filled with water.
• lamp performance
• cleanliness of flow cell
Agilent 1200 Series LC – Agilent Restricted
Page 43
2/18/2010
Dark Current Test
Measures the detector noise (electronics)
while the lamp is off (dark current)
=> checks integrity of sample and reference photodiode, sample and
reference VWA boards and VWM ((main)) board
Digital/Analog Converter (DAC) Test
Converts a test voltage of 10 µV to an analog signal, which should have a
noise below 3 µV, otherwise D/A converter on VWD main board is defect
(requires board replacement)
Agilent 1200 Series LC – Agilent Restricted
Page 44
2/18/2010
VWD Grating Motor Test
Checks the motor position steps for grating
movement. To be done if calibration or holmium filter tests fail.
Caused by defective grating motor, grating position sensor of
VWD main board
VWD Filter Motor Test
Checks
Ch
k the
h motor off the
h second
d order
d cut-off
ff filter.
fil
movement. To be done if holmium filter tests fails.
Caused by defective filter motor
motor, filter position sensor of VWD
main board
Agilent 1200 Series LC – Agilent Restricted
Page 45
2/18/2010
VWD scan
During a method execution, a sample spectrum can be
acquired Therefore two scans are needed: a solvent/blank scan
acquired.
and a sample scan. The online spectrum is displayed as the
difference of both scans. The flow needs to be stopped
(flow = 0) during the sample scan.
Agilent 1200 Series LC – Agilent Restricted
Page 46
2/18/2010
Flow Cells
Standard and Micro
• Maximum pressure 40 Bar
Materials: SST, quartz, PTFE
Standard cell: 10mm, 14ul
Micro cell: 5mm,, 1ul
Gasket #2 has different
aperture
High Pressure
Maximum pressure 400 Bar
Materials: SST, quartz, Kapton
10mm, 14ul
High Pressure Cell
Standard and Micro Cell
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Diode Array Detector (DAD) principle
I0
I
Measurement of I0?
Agilent 1200 Series LC – Agilent Restricted
Page 48
2/18/2010
DAD settings: slit width
smaller slits ☺ increase the spectral resolution and
noise due to lower intensity
Agilent 1200 Series LC – Agilent Restricted
Page 49
2/18/2010
DAD settings: peak width/
response time/time resolution
smaller peak widths
☺ increase time resolution
☺ improves separation of peaks
increase noise
increase amount of data
Agilent 1200 Series LC – Agilent Restricted
Page 50
2/18/2010
DAD diagnostic tests
• filter
filt ttestt
• intensity test
• dark-current
dark current test
• slit test: checks intensity change when changing slit widths
• holmium oxide test
• spectral flatness test: maximum noise in spectrum < 2 mAU
• ASTM noise test: noise over 20 minutes without flow cell < 0.02 mAU after
1 hour of equilibration
• cell test: ratio of intensity in full range with and without cell
• wavelength verification and re-calibration based on α and β emission lines
of deuterium lamp
• test chromatogram
Agilent 1200 Series LC – Agilent Restricted
Page 51
2/18/2010
DAD intensity test
Agilent 1200 Series LC – Agilent Restricted
Page 52
2/18/2010
DAD flow cells
Typical Column
Length
Typical Peak
Width
<= 5 cm
0 025
0.025
10 cm
0.05
20 cm
0.1
>= 40 cm
0.2
Recommended Flow Cell
500 nl Flow Cell
Semimicro Flow
Cell
High-Pressure
Flow Cell
Standard Flow
Cell
Typical Flow Rate
0.01-0.2 mL/min
0.2-0.4 mL/min
0.4-0.8 mL/min
1-2 mL/min
Internal Column Diameter
0.3-1
0.3
1 mm
2.1 mm
3.0 mm
4.6 mm
0.05-5 mL/min
Agilent 1200 Series LC – Agilent Restricted
Page 53
2/18/2010
Flow Cells
Standard and Semi-micro
Maximum pressure 120 Bar
M t i l SST,
Materials:
SST quartz,Teflon
t T fl
Apertures of front and rear
gaskets have different diameters
Standard cell uses cell body for
additional
dditi
l heat
h t exchange
h
Semi-micro cell: 6mm, 5uL
Standard: 10mm, 13uL
Micro
Maximum pressure 400 Bar
M t i l SST,
Materials:
SST quartz,
t Kapton
K t
6mm, 1.7ul
Mi
Micro
C
Cellll
Different apertures
Standard and Semi
Semi-Micro
Micro Cell
Group/Presentation Title
Agilent Restricted
Month ##, 200X