mantenimiento y resolución de problemas habituales en … · 2016-09-03 · octubre 2013. 2...
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Mantenimiento y Resolución de Problemas habituales en Cromatografía UHPLC - GC
Manuel Otero
Field Service Engineer
Especialista en UHPLC, GC y Aplicaciones
MadridOctubre 2013
2
Mantenimiento y Resolución de Problemas habituales en Cromatografía
UHPLC GC
3
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
4
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
5
Agilent UHPLC 1290 Series
Binary pump with Built-in degasser
• Best in class for analytical
and UHPLC applications
• 1200 bar
• Binary or quaternary pump
• Vials and microliter plates
• Up to 100°C column compartment
• DAD at 160 Hz
• Stack configuration different.
– More stability – heavier modules on
the bottom.
– Shortest possible connections.
– Requires longer inlet tubing from
solvent reservoirs.
– Tubing clips route connection tubing.
Detector
Column Compartment
Autosampler
6
Capillary Connections
0.12 mm SST 340 mm
0.12 mm SST 340 mm
0.12 mm SST 340 mm
Flo
w
Flo
w
Tubing diameters depend on delay volume configuration -
from 0.12 to 0.17 mm i.d.
Agilent 1290 Agilent 1260
7
Stack Configurations Agilent 1290
8
Mobile Phase Recommendations
• Contaminated solvents or microbial growth in the solvent
bottle may plug the solvent inlet filter, reducing pump
performance.
– If possible, use sterile solvent bottles.– Filter solvents through sterile filters (< 0.2 µm).
– Replace the solvents every two days or refilter.
– Avoid exposure to direct sunlight or use brown glass bottles.
– Consider adding 0.1 to 1 mM sodium azide or 5-10% organic to the aqueous mobile phase to inhibit microbial growth.
9
pH Considerations
• In general, Agilent Infinity modules operate over a pH range 1
to 12.5.
• pH < 2.3– Solvents must not contain acids that attack stainless steel.
• pH > 9.5– Replace standard (Vespel) rotor seals in all rotary valves with
either Tefzel or PEEK seals.– Replace the standard glass solvent inlet filters with stainless steel
inlet filters.– Be aware that quartz flow cell windows are slowly etched. Do not
let high pH solvents stand in the flow cell for long periods.
10
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
11
Maintenance of Degassers
• Clean the degasser lines by flushing with isopropanol.
• When using buffers, flush with water, then with isopropanol.
• Check for air bubbles in outlet lines.
• Be aware of the possibility of microbial growth in aqueous
phases.
• Unused channels should be left in isopropanol.
• May have to exchange the vacuum pump, sensor, solenoid
valve, or vacuum chamber – standard and microdegassers.
• Integrated degassers require unit replacement with
malfunction.
12
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
13
Agilent 1290 Solvent Delivery System
A1 B1 B2 A2
Degasser
Pump Head BPump Head A
Jet Weaver
Purge Valve
Waste
Inlet Valve
Pump SealOutlet Valve
Piston
High Pressure Filter Assembly
Pressure Sensor
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Replace Pump Head Agilent 1290 Binary or Quaternary Pumps
Replace pump heads with refurbished,100% tested pump heads. Includes thefilter, pump seals, pistons and inlet andoutlet valves.
Disassembly is discouraged. A kit with the correct tools and partsmust be purchased and used.
Exchange Complete Head
Remember to use Lab Advisor to prepare thePump head for removal Tools > Install/Remove Pump Head. Requires a torque hex key.
15
Agilent 1290 High Pressure Filter Assembly
01018-22707 PTFE frit (pack of 5)5001-3707 Gold seal, outlet5042-1346 Cap
Filter Frit
Gold Seal
CapThe filter frit in the outletvalve should be replaced regularly depending on the system usage.
Filter frit
Binary Pump
Quaternary Pump
16
System Pressure Test
• Determines the leak rate of the system between the pump outlet valves and a blank nut.
• The blank nut can be positioned at different locations before the detector flow cell.
• Perform the test at the normal operating pressure of the system.
17
Pump Head Leak Test (1290)
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Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
19Page 19
20 µl loop capillary (in box)
20 µl analytical Head
needle seat40 ppm carryover
blocked needle seat for diagnostics
1200 bar injection valve
Agilent 1290 High Performance Autosampler
Procedure Frequency
Change Needle/Needle
Seat 60,000 needle into seat
Change Metering Seal 30,000 injections
Peristaltic Pump
Cartridge 3000 hours
Change Rotor Seal 30,000 injections
20
Replace Needle – Agilent 1290 Autosampler
1. Turn the needle carrier 90°clockwise.2. Flip the leak guide open.3. Hold the needle assembly in position and
loosen the loop fitting capillary.
4. Pinch the holder clamp, pull back and remove the loop capillary from the needle assembly.
5. Push the silicon safety tube over the needle.6. Insert the loop capillary into the needle
assembly and tighten by hand.
7. Pinch the holder clamp and reinsert theneedle assembly into the needle carrier.
8. Tighten the fitting.
21
Isolation seal
Rotor seal
Replacing the Rotor Seal - I
1. Remove all capillaries.
2. Remove stator screws.
3. Remove stator head, stator face and stator ring
4. Remove defective rotor seal
5. Reinstall isolation seal (spring towards the valve body)
6. Reinstall the rotor seal (grooves facing outward)
Stator head
Stator face
Stator ring
22
Replacing the Rotor Seal - II
7. Install the stator ring (note the
orientation).
9. Replace stator screws and tighten
alternately, two turns at a time.
From Pump
To column
To needle seatWaste
Plug
To metering device
10. Reconnect all capillaries.
8. Place the stator face on the stator head.
Install them together onto the valve.
23
Tests for Agilent Infinity 1290 Autosampler
Agilent 1290 Autosampler Tests
System Pressure test
Sampler Leak Test
Sample transport Self Alignment
ChemStation
Temperature Mainboard
1. In Lab Advisor, Select Tests.
2. Click on module for availabletests.
24
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
25
Thermostat Function Test
Tests heating and cooling performance.
26
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
27
Optical design of Agilent 1260 and 1290 Diode Array Detector
Deuterium
Lamp
Max-Light Cartridge Cell
Programmable
or Fixed
Slit
Diode Array
GratingMirror
10mm and a 60mm path length cells are available
28
Replacing the Deuterium Lamp 1290 DAD
5190-0917
Unscrew the three screwsto remove the cover.
Disconnect the lamp, remove andreplace.Do not touch theGlass bulb withyour fingers.
29
Cleaning the Max-Light Cartridge
If there are low counts on the Intensity Test or the Cell Test
1. Flush the flow cell with isopropanol or ethanol for some time.
2. Remove the cell from the cartridge holder.
3. Carefully clean the light inlet and outlet using lens tissue or Q-tips dipped in alcohol.
If alcohol cleaning fails, you can try the cell cleaning fluid (5062-8529) or replace the cartridge.
30
Wavelength Calibration
Uses the zero-order position, the
656 nm (alpha-emission line) and
the 486 nm (beta-emission line)
to calibrate the detector.
31
� Measures the intensity of the lamp.
� 4 spectral regions used to evaluate
the complete wavelength range.
Intensity Test
Reasons for Test Failure
- Absorbing solvent or air bubble in
the cell.
- Dirty or contaminated flow cell.
- Contaminated optical components.
- Old lamp.
32
Cell Test Check for dirty or contaminated flow cell.
33
Mantenimiento UHPLC
� Introducción del Agilent 1290 Infinity Series
�Desgasificador
�Bomba
� Inyector Automático
�Compartimento de Columnas
�DAD
�Problemas de Ejemplo
34
Pressure problems.
Pressure too high. Pressure too low.Pressure Unstable.
Problems with the System Pressure
35
Pressure Problem One
Pressure Too
High.
• Column inlet frit contaminated.
• Frit in purge valve contaminated.
• Column contaminated.
• Blockage in a capillary, particularly needle
seat capillary.
• Rotor in injection valve plugged.
• Injection needle or needle seat plugged.
Pressure Measurement.
Use this valve to
divide the
system.
36
Pressure Problem Two
Pressure Too Low.
� Solvent inlet frit plugged.
� Leak in a capillary connection or
other part (pump seals).
� Wrong solvent or flow rate.
� Inlet valve defective.
� Multichannel Gradient valve
incorrectly proportioning.
� Outlet ball valve defective.
� Column defective
(stationary phase).
Pressure Measurement.
Solvent inlet
frits.
37
Pressure Problem Three
Pressure Fluctuation.
• Solvent inlet frits plugged.
• Solvent not degassed.
• Pump seals leaking.
• Outlet ball valve defective.
• Active inlet valve defective.
Usually an indication there is air in the pump.
Solvent inlet
frits.
38
Reproducibility
•Area and Peak Height problems together point to the autosampler system.
•Area and Retention Time problems together point to the pump.
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Rotor seal.
Metering device seal.
Pump.
Column.
Problems with Reproducibility – Peak Areas
Peak Areas not
Reproducible.
With peak height:• Rotor seal cross-port leak or injection valve not tight.• Metering device seal leaking.• Needle partially blocked.With retention time:• Variable pump flow rate.Other:• Capillary from injector to detector not tight.• Detector equilibration problems.
40
Problems with Reproducibiliy – Retention Time
Retention Times not Reproducible.
Pump Problems:
• Mobile phase composition problems.
• AIV, outlet ball valve defective.
• Flow rate problems.
Column Oven Problems:
• Temperature fluctuations.
Other:
• Column equilibration.
• Column deterioration.
41
Possible Causes:
• Dirty Flow Cell.
• Dirty mobile phase.
• Detector Lamp Failing.
• Pulses from Pump (if Periodic).
• Temperature Effects on Detector (RI).
• Air Bubbles passing through Detector.
• Gradient elution.
• Immiscible Solvents.
Baseline Fluctuations
42
Baseline Noise
Baseline Noise
Stop Flow
Noise PersistsDetectorPump/Mobile
Phase
YES NO
Other Questions to Ask
• Have you changed your mobile phase
composition?
• Have you changed your acquisition
wavelength?
• What mobile phase was last used in
your instrument?
• Do you have a miscibility problem?
• Are your solvents dirty?
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Mantenimiento y Resolución de Problemas habituales en Cromatografía
UHPLC GC
44
Mantenimiento GC
� Introducción del Agilent GC7890B
� Inlets
�Columnas
�Detectores
45
Mantenimiento GC
� Introducción del Agilent GC7890B
� Inlets
�Columnas
�Detectores
46
Agilent 7890B Top View
Injector
Gripper arm
Tray
Turret
47
Agilent 7890 Front View
48
Agilent 7890 Rear View
49
Gas Supply Characteristics
Gases must be chosen with consideration of the type
of detector used:
• Inert.
• Dry.
• Pure.
• 99.9995% pure.
Using Compressed Gases Safely
Obtain safety information from your company's safety department or from your local gas supplier.
50
Gas Traps and Filters
• Molecular Sieve Trap (moisture trap).
• Oxygen trap.
• Hydrocarbon filter.
• Purification systems.
51
Gas System Controls
• Supply gases must be regulated to provide a constant
pressure to the GC flow control systems.
– Use two-stage pressure regulators on cylinders.
– Pressure regulator requires 10-15 psi pressure
differential to control properly.
• Set proper supply pressures:
– Carrier, 4.0-7.0 bar. (Recomendado 5.0 bar
– Air, 4.0-5.5 bar.
– Hydrogen, 3.5-5.0 bar.
• Individual on/off valves can be used for convenient
isolation of supply lines or instruments.
• In-line regulators and gauges can be used for individual
control/monitoring of pressures to instrument groups.
52
Mantenimiento GC
� Introducción del Agilent GC7890B
� Inlets
�Columnas
�Detectores
53
Inlet Types
• Split/Splitless inlet (SSI).
• Purged-packed inlet (PPI).
• Programmable cool on-column inlet (PCOC).
• Multi Mode Inlet (MMI).
• Volatiles inlet (VI).
• Valves (sample introduction only).
Inlet pressure/flow controls:
• Electronic Pressure Control (EPC).
54
Agilent 7890 Inlets Overview
Inlet Column Mode
Sample
Concentration Comments
Sample
To Column
Split/Splitless Capillary Split High Very little
Pulsed Split High Useful with large
injection
Very little
Splitless Low All
Pulsed Splitless Low Useful with large
injection
All
COC Capillary n/a Low or labile Minimal
discrimination
and
decomposition
All
PPI Packed n/a Any All
Large Capillary n/a Any OK if resolution not
critical
All
MMI Capillary Split High Very little
Pulsed Split High Very little
Splitless Low All
Pulsed Splitless Low All
Solvent vent Low Multiple injections Most
VI Capillary Direct Low Lowest dead volume All
Split High Very little
Splitless Low All
55
S/SL Assembly
56
S/SL Seals
5188-5367 Gold-plated seal
5182-9652 Gold-plated (cross) seal
18740-20880 Stainless steel seal
5061 - 5869 Washer
Thermal nut
Seal
Washer
Reducing nut
Gold seal (original)
Gold seal (cross)
57
Liners
Split
Splitless
58
S/SL Pressure Decay Test
• Set Inlet Temp to 70.0 ºC.
• Cap off inlet column fitting.
• Set septum purge flow to 30 mL/min.
• Set inlet to Split Mode.
• Set Total flow to 60 mL/min.
• Set inlet pressure to 25 psi.
• Allow pressure to stabilize.
• Cap the septum purge line with TCD detector
plug.
• Measured Flow at Split Vent = 60 mL/min.
• Close inlet gas valve.
• Set inlet pressure to Off.
• Monitor “actual” pressure value for 5 minutes.
• < 2.0 psi decrease indicates no leak.
59
S/SL Pressure Decay Test (con’t)
60
Routine Injection Port Maintenance
• Change septum.
• Clean or replace syringe.
• Perform pressure decay or leak test and repair any leaks.
• Clean or replace liners/inserts.
• Replace O-rings.
• Clean or replace seal and washer (SSI).
61
Mantenimiento GC
� Introducción del Agilent GC7890B
� Inlets
�Columnas
�Detectores
62
Typical Chromatographic Problems
• No peaks.
• Poor response.
• One or more peaks missing.
• Tailing peaks.
• Column deterioration caused by contaminated carrier gas.
63
No Peaks
• Leaks.
• Flow controller operation.
• Damaged or faulty syringe.
• Detector or integrator problems.
64
Poor Response
• Adsorption of the sample by column materials.
• Loss of liquid phase (column too old).
• Sample size too small.
• Defective syringe.
• Poor injection technique.
• Leaks.
• Incorrect injection port or column temperature.
• Incorrect detector sensitivity.
65
One or More Peaks Missing
• Sample too dilute.
• Adsorption by column tubing, packing, and so on.
• Incorrect injection port or column temperature.
• Incorrect flow rates.
• Leaks.
66
Tailing Peaks
• Adsorption of sample by column materials.
• Poor column packing technique.
67
Deterioration by Contaminated Carrier Gas
• Water or oxygen contamination may depolymerize stationary
phases like carbowax, FFAP, and SP1000.
68
Cross-linked or bonded phases are resistant to damagefrom water or organic solvents.
Factors that Cause Column Damage
• Exceeding column upper temperature limits (especially
damaging to polar stationary phases).
• Exposure to oxygen at elevated temperatures.
• Exposure to inorganic acids or bases.
• Matrix contamination from poor gas quality or inadequate
sample preparation.
• Aqueous samples used with polar stationary phases.
69
Mantenimiento GC
� Introducción del Agilent GC7890B
� Inlets
�Columnas
�Detectores
70
Description of Detectors
Molecules are energized by a plasma source and separated into excited atoms. As electrons
return to their stable state, they emit light, which is element specific.
AED
Molecules absorb infrared energy, the frequencies of which are characteristic of the bonds within
that molecule.
IRD
Molecules are bombarded with electrons producing ion fragments, which pass into the
spectrometer’s mass filter. The ions are filtered based on their mass/charge ratio.
MSD
Molecules are ionized by excitation with photons from a UV lamp. The charged particles are
then collected, producing a current.
PID
Halogens, sulphur, or nitrogen compounds are mixed with a reaction gas in a reaction tube. The
products are mixed with a suitable liquid, which produces a conductive solution.
ELCD
Sulphur and phosphorous compounds burn in a flame, producing chemiluminescent species,
which are monitored at selective wave lengths.
FPD
Nitrogen and phosphorous compounds produce increased currents in a flame enriched with
vaporized alkali metal salt.
NPD
As electronegative species pass through the detector, they capture low energy thermal electrons,
causing a decrease in cell current.
ECD
Components burn in a flame, producing ions, which are collected and converted into a current.FID
Filament temperature increases as analytes present in the carrier gas pass over it, causing the
resistance to increase.
TCD
Brief Description
Types of
Detector
71
Exploded Parts View of the FID
72
FID Detector Cutaway
CollectorInsulators
Jet
Column nut
Igniter
Air
H2 and Make-Up gas
73
Agilent 7890 FID Jets
Capillary-Optimized FID Jets
Jet Type Part# Jet Tip ID
Capillary G1531-805600.29 mm
0.011 in.
High Temp G1531-806200.47 mm
0.018 in.
Adaptable FID Jets
Jet Type Part# Jet Tip ID
Capillary 19244-805600.29 mm
0.011 in.
Packed 18710-201190.47 mm
0.018 in.
Paced Wide Bore 18789-800700.79 mm
0.030 in.
High Temp G1531-806200.47 mm
0.018 in.
74
Agilent 7890 FID Setup – Column Installation
mm
01
02
03
04
05
0
mm
01
02
03
04
05
06
07
0
48mm68mm 1mm
General Rule:
Push to tip of Jet
then withdraw 1 mm.
Capillary Optimized FID Adaptable FID
75
FID Parameters
FRONT DET (FID)
Temp 250 250 <
H2 flow 30.0 30.0
Air flow 350.0 350.0
Mkup 15.0 15.0
Flame Off
Output 0.0
CONFIGURE FRONT DET
Mkup gas type N2 <
Lit offset 2.0
Electrometer On
76
Agilent 7890 Flame Ionization Detector
• Typical Problems:
– Flame blowing out or not lighting.
– Spiking.
– Low Sensitivity.
– Noise.
– Drift.
77
Solving FID Lighting Problems
• Check detector parameter settings (keyboard):– Flows.– Flame On.– Detector On.– Lit Offset.
• Check jet.
• Check igniter.
• Check column connections.
• Check gas supply pressures.
• Check solvent and injection size.
78
Solving FID Noise Problems
Turn off
H2 and Air.
Still
Noisy?
Check/Clean/Replace:
•Filters, traps, gases.
•FID jet.
•Column and connections.
•Inlet and consumables.
Check/Clean/Replace:
•Interconnect/collector connection.
•Collector and insulators.
•Detector interconnect.
•Detector board.
NoYes
Electrical Problem. Contamination Problem.
79
Routine FID Maintenance
• Monitor the background signal.
• Check pressures/flows.
• Clean or replace the jet.
• Inspect the igniter assembly.
• Clean the collector assembly.
• Remove, trim, and reinstall column.
80
Agilent 7890 Flame Lighting Problems
• Measure flows.
• Clean/replace jet.
• Are column and fittings tight?
• Do we have supply gases?
• Is detector on?
81
Graciaspor su atención
Manuel OteroAGILENT [email protected]
Mantenimiento y Resolución de Problemas habituales en Cromatografía UHPLC - GC