Tips and Tricks of LC User Maintenance

Ad d T i TiAdvanced Topic: Tips and Tricks of LC User

MaintenanceMaintenance

Edward KimApplication Engineer

February 19 2010February 19, 2010

Agilent 1200 Series LC Agilent Restricted

2/18/2010Page 1

Troubleshooting in HPLC

20001500001000500

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Agilent 1100/1200 Series System Operating PrinciplesOperating Principles

Pumping principlePumping principle

Sampler principles

TCC principle

Detector principle


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Fast Serviceability Still Unmatched

Savings from E-PAC Design:

f60% reduction in mechanical partsf90% reduction in screw jointsf90% reduction in screw jointsf70% reduction in assembly timef90% reduction in disassembly

time

E-PAC: Expanded Polypropylene foam

All pure materials for low cost recycling


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Agilent 1100/1200 Module Serial Number FormatSerial Number Format

10 characters:

DE 91602274

I di id l d l tWhere made? Date code

(16st week of 1999)

Individual module counter (how many have been made?)

JP = JapanDE = GermanyUS = USA

Each module has it's own module counter. The date code marks a major change in production

US = USACN = ChinaFR = France

The date code marks a major change in 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.


2/18/2010Page 5

lower 55000 to 1100Series.

Degassers - Overview

G1322A Vacuum Degasser (aka Standard Degasser/Online Degasser)

G1379A Micro Vacuum Degasser 1100 Series (obsolete)G1379A Micro Vacuum Degasser 1100 Series (obsolete)

G1379B Micro Vacuum Degasser 1200 Series


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Standard degasser operating principle


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Avoid growth of algae


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Comparison of degassers

G1322A:

better for applications using methanol 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


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Maintenance Cleaning Flush the solvent tubes withCleaning Flush the solvent tubes withisopropanol.

When using buffers, flush with water.

M h t h th May have to exchange the vacuum pump, sensor, solenoid valve, or chamber.

Replace solvent inlet frits

Solvent

A B C D

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Pump

Pumps - Overview

G1310A I ti P l t ( i ht b i t )G1310A Isocratic Pump: one solvent (might be mixture)

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]


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Isocratic pump


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Quaternary/binary pump

Quaternary Pump Binary Pump

high pressuremixing

multi-channel gradient valve (MCGV)

l i i


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low pressure mixing

Pump options

Solvent Selection Valve (P/N G1312-60000)Solvent Selection Valve (P/N G1312 60000)

Normal Phase Seals (P/N 0905-1420)

Seal Wash (P/N 01018 68722) Seal Wash (P/N 01018-68722)

Active Seal Wash (P/N G1311-68711)


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Seal wash option

Seal Wash, active/passive


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System Pressure Test

Pressure tightness up to 400 bar of the system (this is no pump test!)Ti ht t ith bl k t f bl t TCCTighten system with blank nut, preferably at TCCUse isopropanole as solventPressure increased to 390 bar, decrease lower than 2 bar/minute


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Leak test for quaternary pump

12

Pressure tightness of the quaternary pumpTighten the pump outlet with a blank nutUse isopropanole as solvent, flush system yprior to test (remove bubbles)

Steps:Ramp 1: both plungers deliver for two strokes (1-2-1-2)

problems at plateau 1 check OBV plunger/seal 2

Plateau 1: plunger 2 with low flow (2 l/min)Ramp 2: Plungers 2 and 1, high flow (153 l/min)Plateau 2: Plunger 1 delivers with low flow (2 l/min)Ramp 3, 4: Plungers 2-1-2-1 at high flow, up to 390 bar

check OBV, plunger/seal 2,purge valve, blanking nut

problems at plateau 2 check AIV, plunger/seal 1


2/18/2010Page 17

tightness of pump? degassing efficiency? also affected by chamber 2

G1312A Leak Test

IF A2 & B2 failed:everything after the both OBV could beleaking including blank nut and purge valve


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g g p g

Pump parametersflow: volume delivered per time range depends on pump typeflow: volume delivered per time, range depends on pump type

composition: percentage for each solvent (bin./quat. pump)

t k th l i f 20 l t 100 l f dstroke: the volume in a range of 20 l to 100 l of compressed solvent delivered by one movement of piston 1a lower stroke improves mixing but decreases lifetime of seals p gand valves

stroke

auto100 l auto: minimum possible

stroke for a chosen flow(recommended setting)

settable stroke

20 l

( g)


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flow0.1 ml/min 5 ml/min

Pump parameters: compressibilityCompressibility: relative volume change per pressure changeCompressibility: relative volume change per pressure change = V/(Vp), e.g. water 46 10-6 bar, acetonitrile 115 10-6 bar, isopropanole 100 10-6 bar, see manual/on-line helpis corrected by initial pump speed for avoiding pressure fluctuations (pressure ripple should be below 1%)


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Using the pressure plot

piston 2 delivers pressure (ripple)

piston 1 deliverspiston 1 delivers

piston 2 delivers

pressure drop


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p p

Optimization of compressibility settings

ibilit tti t hi hcompressibility setting too high

compressibility setting too low

compressibility changes during gradients = changing composition prior to mixing for quaternary pumps


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prior to mixing for quaternary pumps

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 toof 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 columncontributes to both the delay and dispersion volume i e peakscontributes to both the delay and dispersion volume, i.e. peaks come later and are broader

=> use short connections and small diameters (columns, ( ,capillaries)


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Delay and dispersion volumesfor binary and quaternary pumpfor binary and quaternary pump

Quaternary pump: 800 l to 1100 l

Binary pump: 180 l to 480 l

Largest contribution by damper (1l/bar)


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Autosamplers - overview

G1313A Autosampler (obsolete)

G1329A Thermostated Standard Autosampler (1100/1200) [G1329B: ALS SL (RRLC) high pressure variant]

G1367A Well Plate Autosampler (1100 Series)G1367A Well-Plate Autosampler (1100 Series)G1367B High-Performance Autosampler (1200 Series),HiP ALS[1367C: HiP ALS SL (RRLC)]

[1367D HiP ALS SL+ (RRLC l l )][1367D: HiP ALS SL+ (RRLC, low carry over als)]

G1330A/B Autosampler Thermostat[special samplers for other applications: micro autosampler micro wellplate sampler dual-loop[special samplers for other applications: micro autosampler, micro wellplate sampler, dual-loop autosampler, prep autosampler CTC]


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Standard ALS Principle

PumpPump PumpMainpass Draw Sample

(Bypass)Inject

to Column to Column to Column


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HiP ALS Principle


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ALS (Std/HiP) Options

Extended Volume Metering Device 900l

MultiDraw Kit 400l & 1400l & (5000l prep)MultiDraw Kit 400l & 1400l & (5000l prep)

Red Needle Seat Capillary 0,12mm ID


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Problems with Reproducibility Peak Areas

Peak Areas not Reproducible

Seal

PumpReproducible Pump

With peak heightRotor seal cross port leak or injection valve not tightRotor seal cross-port leak or injection valve not tightPiston seal of metering unit leakingNeedle partially blockedWith retention time

Column

With retention timeVariable pump flow rateOtherCapillary from injector to detector not tight

Rotor Seal p y j g

Detector equilibration problems

Page 29

Thermostatted Column Compartment- overviewoverview

Th t tt d C l C t t G1316A TCCThermostatted Column Compartment G1316A TCC [G1316B: TCC SL (RRLC)]

[G1316C: TCC SL+ (Method Devellopment Solution)][G1316C: TCC SL+ (Method Devellopment Solution)]


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TCC principle

G1316A


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Agilent 1100/1200 Series ThermostattedColumn CompartmentColumn Compartment

G1316A

Options:

Column Switching Valve 6 or 10 PortColumn Switching Valve 6 or 10 Port

SpecsSpecs:

10 degrees below ambient to 80 deg C

up to 80 deg C: max flow rate 5ml/min


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Agilent Capillary Color Code

C l i dColor i.d.

Red 0.12 mm

Blue 0.25 mm

Green 0.17 mm

Note: All fittings in the 1100 HPLC are Swagelok compatible, EXCEPT the ports on model 77251 Rheodyne valve.

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77251 Rheodyne valve.

Fittingsg

0 090 i 0 130 i

Swagelok Waters

0.090 in. 0.130 in.

Parker Rheodyne

0.090 in.0.170 in.

Valco Uptight

0.090 in.0.080 in.

Troubleshooting LC Fittings Part II

34

Troubleshooting LC Fittings Part II,J.W. Dolan and P. Upchurch, LC/GC Magazine 6:788 (1988)

Detectors

G1314A Variable Wavelength Detector (1100 Series) VWD obs.

G1314B Variable Wavelength Detector (1200 Series) VWDG1314B 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 Array Detector (1100/1200 Series) DAD SL]G1315D Diode-Array Detector (1100/1200 Series) DAD SL]

G1365B Multiple-Wavelength Detector (1100/1200) MWD[G1365C/D Multiple-Wavelength Detector SL (1200 Series) MWD SL[ p g ( )

G1321A Fluorescence Detector (1100/1200 Series) FLD

G1362A Refractive Index Detector (1100/1200 Series) RID

G4218A Evaporative Light Scattering Detector (1200 Series) ELSD]


2/18/2010Page 35

G4218A Evaporative Light Scattering Detector (1200 Series) ELSD]

Lamp MatrixDetector Original Secondary VIS Lamp

New

Detector Original Secondary VIS LampDAD G1315A/B/C

2140-0813 5181-1530(2140-0820) G1103-60001MWD G1365A/B/CDAD SL G1315D/E

2140-0820 5181-1530(2140-0813) G1103-60001MWD SL G1365D/E1290 Infinity G4212A (New) 5190-0917 NA G1103-60001VWD G1314A/B/C G1314-60100 2140-0813 G1103-60001VWD SL+ G1314D/E G1314-60101 2140-0820 G1103-60001UV VIS 8453A 2140-0605 2140-0813 G1103-60001UV VIS 8453A 2140-0605 2140-0813 G1103-60001CE G1600A 2140-0585 NA NACE G7100A (New) 5190-0917 NA NA

New SL and SL+ detectors shipped with RFID lampSL 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 changedThe UV-VIS system can run with a long life lamp. The CE system can not.

Group/Presentation TitleAgilent Restricted

Month ##, 200XPage 36

Lamps with RFID chip can be used with old detectors, but no functionality of the chip.

VWD principle

I0

I

I0


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VWD flow cells

Typical Column Length

Typical Peak Width

= 40 cm 0.2

0.05-0.2 mL/min 0.2-0.4 mL/min 0.4-0.8 mL/min 1-2 mL/min 0.05-5 mL/min

1.0 mm 2.1 mm 3.0 mm 4.6 mm

Typical Flow Rate

Internal Column Diameter

For Pressure Above 100 bar


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Baseline stabilization

VWD baseline shows a slight drift for the first 60 minutes:g


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VWD diagnostic tests


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Wavelength calibration

Or: how to calibrate the rotation angle of the grating

00

1

1

656 nm

> T iti d d d d fi t d 656 i i li=> Two positions needed: zero order and first order 656 nm emission line of deuterium lamp

required after repair, part replacements or for PM


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the 656 nm position is checked each time the lamp is switched on

VWD: Holmium oxide test

Similar to wavelength calibration, done for 3 absorption lines of holmium filterVerifies success of wavelength calibration, used in OQ/PV and after flow cell maintenance/repair


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VWD intensity testMeasures intensity for complete wavelengthMeasures intensity for complete wavelength range and for a flow cell filled with water.

lamp performance

cleanliness of flow cell


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Dark Current TestMeasures 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 V to an analog signal, which should have a noise below 3 V, otherwise D/A converter on VWD main board is defect (requires board replacement)


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(requires board replacement)

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

Ch k h f h d d ff filChecks the motor of the second order cut-off filter.movement. To be done if holmium filter tests fails.

Caused by defective filter motor filter position sensor of VWDCaused by defective filter motor, filter position sensor of VWD main board


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VWD scan

During a method execution, a sample spectrum can be acquired Therefore two scans are needed: a solvent/blank scanacquired. Therefore two scans are needed: a solvent/blank scan 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.


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Flow CellsHigh PressureStandard and Micro

Maximum pressure 400 Bar

Materials: SST, quartz, Kapton

10mm, 14ul

Maximum pressure 40 Barz Materials: SST, quartz, PTFEz Standard cell: 10mm, 14ulz Micro cell: 5mm, 1ul,z Gasket #2 has different

aperture High Pressure Cell

Standard and Micro Cell

Group/Presentation TitleAgilent RestrictedMonth ##, 200X

Diode Array Detector (DAD) principle

I0

I

Measurement of I0?


2/18/2010Page 48

DAD settings: slit width

smaller slits increase the spectral resolution and / noise due to lower intensity


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smaller slits increase the spectral resolution and / noise due to lower intensity

DAD settings: peak width/response time/time resolutionresponse time/time resolution

smaller peak widths increase time resolution improves separation of peaks improves separation of peaks/ increase noise/ increase amount of data


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DAD diagnostic testsfilt t t filter test

intensity test

dark current test dark-current test

slit test: checks intensity change when changing slit widths

holmium oxide testholmium 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 and emission lines of deuterium lamp

test chromatogram


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test chromatogram

DAD intensity test


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DAD flow cells

Typical Column Length

Typical Peak Width

Flow CellsMicro

Maximum pressure 400 Bar

M t i l SST t K t

Standard and Semi-micro

z Maximum pressure 120 BarM t i l SST t T fl Materials: SST, quartz, Kapton

6mm, 1.7ul

z Materials: SST, quartz,Teflonz Apertures of front and rear

gaskets have different diametersz Standard cell uses cell body for

dditi l h t hadditional heat exchangez Semi-micro cell: 6mm, 5uLz Standard: 10mm, 13uL

Mi C llMicro CellDifferent apertures

Standard and Semi-Micro Cell

Group/Presentation TitleAgilent Restricted

Month ##, 200X

Standard and Semi Micro Cell

Tips and Tricks of LC User Maintenance

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