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

14

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.

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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

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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.

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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.

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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.

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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.

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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?

43

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).

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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.

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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?

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Graciaspor su atención

Manuel OteroAGILENT TECHNOLOGIESmanuel_otero@agilent.com

Mantenimiento y Resolución de Problemas habituales en Cromatografía UHPLC - GC