tips and tricks of lc user maintenance...tips and tricks of lc user maintenance presented by greg...

Tips and Tricks

of LC User

Maintenance

Presented by Greg Hunlen, John Palmer and Sue D’Antonio

Goals for this presentation:

1. Introduce the major considerations in LC user maintenance.

2. Cover the primary preventative maintenance tasks that the LC user

needs to be concerned with and plan for.

3. Highlight the key consumables that need to be replaced by the LC

user on a regular basis on any HPLC to maintain performance.

Nano LC Capillary

LC Analytical

LC

Prep LC Chip LC

General Considerations in LC User Maintenance:

Know your:

LC Application and Method

LC System

Sample

Plan for:

Routine Maintenance

Minor (daily/weekly)

Major (6 or 12 month frequency)

Tip #1 – Know all of Your Resources on Agilent

1260, 1200 and 1100 LC Maintenance:

Many available sources of information, including:

Reference Manual – included with each module, .pdf’s on web

1200 LC Maintenance Guide – included with system, .pdf on web, copies available today

1200 LC Maintenance Video’s – available on web,

1200 LC PM (Preventative Maintenance) Checklist – provided at PM delivery

Agilent HPLC Troubleshooting and Maintenance Course (H8969A, 3 days), register on web

1200 LC Diagnostics Software, with Early Maintenance Feedback (EMF)

Your local Field Support Engineer

www. agilent.com/chem

Agilent Support Line, 800-227-9770 opt 4 for applications help

Agilent LC and LC/MS Library: •5990-4957EN LC/LCMS

maintenance guide

•Covers all of the

1100/1200/1260/1290

UHPLC/HPLC modules.

•Select the right Agilent LC

supplies.

•5990-7595EN

•Overview of HPLC

and Method

Development

http://www.chem.agilent.com/en-US/Training-Events/eSeminars/Pages/default.aspx

The Agilent Service Advantage

Agilent Service Guarantee—the most comprehensive guarantee in the industry

Highly Trained Field Service Engineers who resolve more than 85% of calls on the first visit

Global Logistics Network with better than 96% on-time delivery performance for service parts

Agilent Remote Advisor proactive support technology to maximize uptime and deliver lab operations reporting

Ranked #1 in Compliance with >100,000 successful qualification performed worldwide

Guaranteed Pass OQ included with the Advantage Silver and Gold plans for confidence in the success of your qualification

98% Customer

Satisfaction with Agilent

Service Engineers

Only with Agilent…

Agilent Remote Advisor

Agilent Liquid Chromatography systemsAgilent Remote Advisor- Assist Open a service request with the click of a mouse and access

Agilent experts for advanced troubleshooting and diagnostics

Agilent Remote Advisor- Report Track instrument operations with full-featured reporting that

includes an inventory list configuration information,

instrument availability and use, as well as real-time

instrument maintenance and qualification status.

Agilent Remote Advisor – Alert Create SMS-text or email alerts, so you’ll be notified when

maintenance thresholds are reached or when your system

shuts down—protecting your throughput and productivity.

Available as part of your Agilent Advantage service and support

agreements:.

New

Infinitely better Diagnostics,

Maintenance, Calibration

The Agilent Lab Advisor

for (U)HPLC & CE

Very helpful in complex diagnostic situations, where the build in

diagnostic tests do not give definite answers.

This are powerful tools for the expert troubleshooter and self-maintainer!

Make use of

With Instrument Control you do not need any bulky Chromatography

Data System to get a full set of controls and freely configurable and

recordable Signal Plots (pressure plot, detectors signals, etc.)!

Lab Advisor Basic provides a

numerical readout of the EMF

values, with an option to reset

the counters.

* Enables usage based maintenance

Lab Advisor Advanced

additionally provides a

graphical presentation

and options to activate

settable limits.

Activate or Deactivate & Customize Early Maintenance Feedback

(EMF) Limits in Advanced Version!

The Lab Advisor User Interface is divided into six main areas. The content of

these areas changes depending on the Screen selected in the Agilent Lab

Advisor software. Title Bar

Information Panel

Application Panel

Action Panel

Status Bar

Global Screens

System Screens

Help Topics

Navigation Panel

UI

The MWiz is an add-on SW embedded into the Agilent Lab Advisor

It will speed up and guide the customers through the complete process

of Agilent LC instrument maintenance (assembly/disassembly, cleaning,

parts exchange, testing) by a structured and easy to use wizard-like

walkthrough.

Descriptive and visualized instructions (animated pdfs/videos) will help

the customer understand and conduct every step along the way.

Result: customized guide, part ordering list, system specific report.

Maintenance Wizard (MWiz)

BreadCrums

Tip # 2 Know your LC System:

Pump

Autosampler

Column Comp.

Detector

Vacuum

Degasser

THE STACK Flow connections in the stack:

Example setup with 0.17mm ID green capillaries

Solvent bottles - degasser:

G1311-60003 (bottle-head assembly, PTFE-

tubings)

Pump -

autosampler: G1312-67305 (SST, green)

Degasser - pump:

G1322-67300 (PTFE-tubings)

Autosampler - column

compartment: G1313-87305 (SST,

green) Column compartment - column:

G1316-87300 (SST, green)

Column -

detector:

DAD G1315-87311 (SST, coated)

VWD 5062-8522 (PEEK)

Detector - waste:

DAD 0890-1713 (PTFE, wide bore)

VWD 5062-8535 (PEEK)

5062-2463 (corrugated waste tubing, reorder

pack)

Agilent Capillary Color Code

Color i.d.

Red 0.12 mm (0.005 inches)

Green 0.17 mm (0.007 inches)

Blue 0.25 mm (0.01 inches)

Orange 0.50 mm (0.02 inches)

Good connections with Agilent capillaries and

fittings

Consider a capillary starter kit, which includes a Cybertool.

Flexible stainless steel capillaries are exclusively offered by Agilent.

Agilent offers a broad range of connecting capillaries and fittings for all Agilent and non-Agilent columns and accessories.

Kits available with 0.12 or 0.17 mm id or with Micro/Nano capillaries.

Use 0.12 mm id for columns with 3 mm id and smaller, 0.17 mm id for 4.6 mm id and larger.

PEEK coated fused silica capillaries for Micro and Nano LC 25, 50, 75 µm id.

PN: 5065-9937 $578

Capillary/Fitting Starter Kit, 0.12 mm i.d. “red” capillary

Part No 5065-9937 Kit Content:

PEEK capillary 0.12 mm i.d., 1.5m

P/N: 0890-1915

Cybertool – “30 in one”

PEEK fittings 1/16”, 10/pk

P/N: 5063-6591

PEEK fittings 1/16”, 10/pk

Colored - P/N: 5065-4426

SS fittings 1/16”, 10/pk

P/N: 5062-2418

Tubing cutter P/N: 8710-1930

Description Volume

[µl]

Qty Part

number

SS capillary, 105x0.12 mm 1.2 4 5021-1820

SS capillary, 150x0.12 mm 1.7 4 5021-1821

SS capillary, 200x0.12 mm 2.3 2 5065-9935

SS capillary, 280x0.12 mm 3.2 2 5021-1822

SS capillary, 400x0.12 mm 4.5 1 5021-1823

Rheotool P/N: 8710-2391

3 ZDV unions, SS

P/N: 5022-2184 (1)

System – Signal Height System volumes – System dispersion

min 0.5 1 1.5 2 2.5

mAU

0

100

200

350

400

550

600 System dispersion optimized

Peak width 0.018 min


Resolution 1.902

min 0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

System dispersion not optimized



Resolution 0.961

310 mAU

380 mAU

Tip #3 - Reduce LC problems by eliminating most

common sources of flow blockage:

Filter, Filter, Filter !!!

Particles leading to blockage can come from sources located both outside

and inside the LC system:

- Solvent, buffer

- Microbial growth in solvent reservoirs

- The Sample

- Wear of LC components – piston seals, autosampler valve, etc.

Debris will either be captured on the column frit (column = expensive) or

in-line filter (inexpensive replaceable frit)

Preventative Maintenance is the Key!!!

Solvent and Buffer Filtration

As internal diameters of connecting

capillaries, particles in the column

and pore sizes of column inlet frits

are getting smaller and smaller,

filtration of mobile phases is

becoming an absolute must.

Use 0.45 um filter for mobile phase

components

PN: 3150-0577

Care of Mobile Phase

Particulates in the LC 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 filters (< 0.45 µm).

• Replace the solvents every two days (do not re-filter).

• Avoid exposure to direct sunlight (use Aluminum foil,

shield from windows) or use brown glass bottles.

• Consider adding 5-10% organic to the aqueous mobile

phase to inhibit microbial growth.

Growth in H2O over 1 week in a clear bottle

Maintenance (G1322A and G1379B)

• 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.

• Check for solvent compatibility.

• Unused channels should be left in isopropanol.

• May have need to exchange the vacuum pump, sensor,

solenoid valve, or vacuum chamber.

Vacuum Degasser Notes (G1322A)

Priming the Degasser:

• Use a syringe when vacuum tubes are empty or

for fast solvent change. Slowly draw solvent

through degasser membrane into syringe.

• Purge the pump at 3-5 mL/min when system has

been turned off for a period of time (overnight,

weekend)

Changing Solvents :

• Make certain that solvents are miscible

• Exchange any inorganic buffer first with pure

water and then with the new solvent

• Use a minimum of 30 mL per channel for solvent

changes

• Check each solvent outlet tube for gas bubbles

Accessory Kit G1322-68705

Syringe 5062-8534

Syringe adapter 9301-1337

Solvent tubing kit (4 tubes degasser to pump)

G1322-67300

Waste tube 5062-2463

Micro Degasser Notes (G1379B)

Priming the Micro Degasser:

• Use the pump at low to moderate speed

• Priming with a syringe is ONLY recommended if

priming with the pump should fail

• Draw slowly when using a syringe

Changing Solvents :

• Make certain that solvents are miscible

• Exchange the inorganic buffer first with pure

water and then with the new solvent

• Use a minimum of 5 mL per channel for solvent

changes

• Check each solvent outlet tube for gas bubbles

Accessory Kit G1379-68705

Mounting tool 0100-1710

Solvent tubing kit (4 tubes degasser to pump)

G1322-67300

Connecting tubing to connect channels in series

for increased performance (2) G1379-68706

Waste tube 5062-2463

Mounting tool (0100-1710)

Agilent 1200 Series On-line Degasser:

Influence on Detector Baseline

no

degassing

Ab

sorb

an

ce

helium degassing

Agilent on-line

degassing

1 2 3 4 5 6 7 8 9

Time (min)

Solvent: methanol; UVsignal: 210 nm

Why Filter the LC Sample ?

Less blocking of capillaries, frits, column inlet

Less wear and tear of injection and switching valves

Less downtime of the instrument

Improved analytical results

See complete listing of available syringe filters on pp 144-149 of 2007/08

Agilent supplies catalog.

Tip #4 - LC Flow Path Filtration Points –

Frits/Sieves to Replace on a regular basis.

Solvent Reservoir

Pump

In-line filters

Normally placed between the autosampler and LC column

Column

Difficult to replace column inlet frit (not recommended)

May be able to reverse column flow and dislodge blockage (flow to waste), but

may not completely restore column performance.

Most manufacturers use 2 um frits for 3 to 5 um particle columns, 0.5 um frits

for sub 2 um particle columns. Match in-line frit to column frit size.

Purge Valve – Exchanging the PTFE Frit

PTFE- frit

Gold Seal

Plastic cap

1. Unscrew the valve using a 14 mm wrench

2. Remove the plastic cap and the gold seal

3. Take out the frit (tweezers)

4. Install a new frit

5. Replace the gold seal and the plastic cap

6. Install the valve

Note: Realign the waste tube in the correct orientation

during installation.

Tip # 5 - Know Your HPLC Flow Path:

Where are the moving parts?

Where can blockages to flow

occur?

Where are the consumables that

need to be replaced on a regular

basis (PM)?

Where can leaks occur ?

What can I do to eliminate, reduce or

anticipate potential problems with the LC ?

Considerations for U/HPLC systems

Gradient Delay or Dwell Volume

Beginning of Mixing

End of Mixing

The volume between the point of mixing of solvents (usually in

the mixing chamber or at the proportioning valves in the liquid

chromatograph) and the head of an LC column.

Extracolumn Volume

The volume between the effective injection point and

the effective detection point, excluding the part of the column

containing the stationary phase. It comprises the volumes

of the injector, connecting lines and frits, and the detector.

It determines the extracolumn effects.

Data Sampling or Aquistion Rate A term referring to the rate of sampling of a detector

output. To characterize a chromatographic peak at least 20-30

data points must be collected. The data acquisition rate, usually

measured in Hertz, defines how many data points per

second are collected while the peak is moving through the

detector.

High Pressure vs. Low Pressure Mixing Principle

Combination and mixing of

mobile phases after the

pump head

Mixing by low-pressure

proportioning valve before

the pump head

Binary pump

High Pressure Mixing

Quaternary pump

Low Pressure Mixing

• Combination and mixing of

mobile phases after the pump

head

• Possible influence of excess

volume on gradient ( flow

accuracy!)

• Combination and mixing by

low-pressure proportioning

valve before the pump head

• No influence of excess volume

on gradient, but on

composition accuracy!

Delay volume Impact of low delay volume

Programmed

gradient step

Delay volume

Low delay

volume

High delay volume

time

response

What happens with a

programmed gradient?

Total delay volume of the system (sum

of capillaries, mixer, cells, valves..)

High delay vol.

Low delay vol.

10 %

90 %

Delay volume Impact of low delay volume

Delay volume ~ 700 μL

min 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

Delay volume ~ 120 μL

min 0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

100

200

300

400

Column: ZORBAX SB-C18 2.1 x 50 mm, 1.8 μm

Flow: 0.42 mL/min

Pump

Important characteristics

Common to isocratic and gradient pumps

Flow accuracy

Flow precision

Pressure pulsation

Gradient pumps only

Delay volume in low and high pressure mixing

Composition accuracy

Composition precision

Influence on...

Retention time and peak area precision (system to system)

Retention time and peak area

precision (within one system)

Baseline noise

Gradient shape and precision


precision (system to system)


precision (within one system)

The good chromatogram isocratic test sample

30% ACN_/_70% H2O

Flow 1.0 ml/min

Wavelength 254

Temperature 40C

5 micro liter injection

Void volume peak

Useful Solvents for Reversed-Phase

Chromatography

Water

Methanol

Acetonitrile

Isopropanol

Dioxane

Tetrahydrofuran

Elution

Strength

Solvent Miscibility Name

Acetic Acid

Acetone

Acetonitrile

Benzene

Butyl Alcohol

Carbon Tetrachloride

Chloroform

Cyclohexane

Cyclopentane

Dichloroethane

Dichloromethane

Dimethylformamide

Dimethyl Sulfoxide

Dioxan

Ethylacetate

Ethyl Alcohol

Di-Ethylether

Heptane

Hexane

Methyl Alcohol

Methylethyl Ketone

I-Octane

Pentane

I-Propyl Alcohol

Tetrachloroethane

Tetrahydrofuran

Toluene

But

yl A

lcoh

ol

I-Pro

pyl A

lcoh

ol

Di-P

ropy

leth

er

Tric

hlor

oeth

ane

Ace

tic A

cid

Ace

tone

Ace

toni

trile

Ben

zene

Car

bon

Tet

Chl

orof

orm

Cyc

lope

ntan

e

Cyc

lohe

xane

Dic

hlor

oeth

ane

CH

Cl

2

2

DM

FD

MSO

Dio

xan

Eth

ylac

etat

eEth

yl A

lcoh

ol

Di-E

thyl

ethe

r

Hep

tane

Hex

ane

Met

hyl A

lcoh

ol

MEK

I-Oct

ane

Pen

tane

2 2

C H

C

l

4

THF

Tolu

ene

Wat

er

Xyl

ene

Trichloroethane

Water

Xylene

Di-Propylether

Immiscible

Miscible

2-Propanol is an

excellent intermediate

solvent

Operating Principle of the Dual Piston Pump

from Solvent Bottle

to Injector

Piston 1

Piston 2

Active inlet valve

Damper (Pressure measurement)

Purge valve

Outlet ball valve

Seals

Agilent HPLC Pump Models - Analytical

Isocratic Pump Quaternary Pump Std Binary Pump

Pump Head – Main Components

Plunger housing

Pump head

Outlet ball valve

Active inlet valve

Purge valve

Pistons Active inlet valve

Seals

Support rings

Purge valve Outlet ball valve

Lock screw

Standard Pump Head – Exploded View

1260 hardware changes

Overall Routine Pump Maintenance

1. Remove and disassemble the

pump head.

2. Remove and clean pistons.

3. Replace piston seals.

4. If seal wash option is installed,

replace wash seals and

gaskets.

5. Inspect the springs.

6. Reassemble pump head and

reinstall.

7. Perform seal wear-in

procedure.

8. Replace PTFE frit in the

purge valve.

9. Clean or replace the outlet

ball valve.

10. Replace the AIV cartridge.

11. Flush with isopropanol.

12. Clean or replace solvent

inlet filters.

13. Clean the leak sensor.

14. Make certain the waste

tube is in place.

15. Test the pump (Pressure

and Leak Test).

- Covered by an annual Agilent LC PM contract -

Pump Seal Wash Option

– Does your system have

this option ?

• Protects your pistons and seals from excessive wear

• Recommended when using aqueous buffer or salt solutions > 0.1M

• 10% MeOH recommended as pump seal wash solvent

Seal Wash

Option

Wash

solvent

Wash

solvent Peristaltic

Pump Pump head

Wash solvent

Most Common Maintenance Areas of HPLC: Moving parts and associated consumables.

Detector cell

Autosampler

Pump

Pump

Autosampler

Uv Detector

Schematic of Injection System

Metering

device

Injection valve

Sample loop

Principle of Operation

Prior to Injection

Valve in Mainpass Position

Draw Sample

Valve in Bypass Position

Injection and Run

Valve in Mainpass Position

Pump

Column Column

Pump Pump

Column

Tip #6 - Overview of Major Autosampler

Maintenance

Injection Valve Metering device Needle/needle

seat

Gripper (finger caps)

• Replace the injection valve rotor

seal

• Replace the needle and the needle

seat

• Check and replace finger caps if

necessary

• Replace the metering unit seal

• Clean the transport rods

• Clean the waste tubing

• Check the leak sensor

• Perform pressure test


Maintenance Consumables and Tools

Rotor seal, Vespel (pH < 9.5) 0100-1853

Rotor seal, Tefzel (pH > 9.5) 0100-1849

Needle assembly G1313-87201

Needle seat assembly G1313-87101

Metering seal 5063-6589

Finger caps (pack of 15) 5063-6506

Piston (Plunger), if necessary 5063-6586

Isolation seal, if necessary 0100-1852

Peristaltic pump cassette 5065-4445 (hALS and hALS SL only)

Wrench ¼ inch 8710-0510

Hexagonal key, 9/64 inch 8710-2394

Hexagonal key, 4 mm 8710-2392

Hexagonal key, 2.5 mm 8710-2412

Rheotool (slotted socket wrench) 8710-2391

Injection Valve Assembly

Tools:

Hexagonal key.....9/64 inch

Wrench.................1/4 inch

Injecton Valve

7

6 5

3 2

1

4

1. Stator screws

2. Stator head

3. Stator face

4. Stator ring

5. Rotor seal ( Vespel or Tefzel )

6. Isolation seal

7. Valve body

Exchanging the Needle/Needle Seat – Standard

Autosampler G1329A.

Parts:

Needle G1313-87201

Needle seat G1313-87101 (0.17 mm i.d.)

or G1313-87103 (0.12 mm i.d.)

Tools:

Wrench ¼ inch

Hexagonal key 2.5 mm

Needle/Needle seat

Autosampler Maintenance Functions

Before beginning needle or needle seat replacement:

Select "Change Needle” in the autosampler maintenance function.

Instant Pilot:

Metering Device Maintenance

Metering device 1. Screws

2. Piston (plunger)

3. Seal support ring

4. Seal

5. Head body

Metering Device Maintenance

Before beginning this procedure, select “Change Piston“ from the ALS

Maintenance Positions.

Instant Pilot:

Tip #7 – add an In-line filter to protect your LC

Column:

Pump

Autosampler

Column Comp.

Detector

Vacuum

Degasser

Add in-line

filter

Tip #8 - Routine Maintenance Procedure – DAD/MWD Uv

Detectors

• Clean the leak sensors

• Check the waste tube

• Exchange the lamp (if necessary)

• Clean the flow cell (if necessary)

• Wavelength calibration

• Holmium Oxide Test

• Intensity test

• Cell test

• Dark current test

• Filter test


Available Diagnostic

Tests

Detector – Signal height Path length

lcI

IA

0

1logA: Absorption

α: Extinction coeffizient

c: Concentration

l: Path length

Influence of Pathlength on Signal Sensitivity

min 3.8 4 4.2 4.4 4.6

mAU

0

10

20

30

40

50

60

70

80

10mm path length

6mm path length

min 0.1 0.2 0.3 0.4 0.5 0

80Hz

PW=0.30sec

40Hz

PW=0.33sec

20Hz

PW=0.42sec

10Hz

PW=0.67sec

5Hz PW=1.24sec

• Sample: Phenone Test Mix

• Column: Zorbax SB-C18, 4.6x30, 1.8um

• Gradient: 50-100% ACN in 0.3min

• Flow cell: 5ul

20Hz versus 80Hz

+ 40% Peak Width

– 40% Peak Capacity

– 30% Resolution

– 70% Apparent Column Efficiency

10Hz versus 80Hz

+ 120% Peak Width

– 120% Peak Capacity

– 90% Resolution

– 260% Apparent Column Efficiency

Benefit of 80Hz Data Acquisition Rate

Peak Width, Resolution and Peak Capacity in Ultra-Fast LC

Determining Peak Apex

Fit highest data point and those on each side

to a quadratic equation, solve for highest point

1.0 1.8 2.6 Retention Time (min)

Determining Peak Apex

1.0 1.8 2.6

Retention Time (min)

Replacing the DAD/MWD Lamps

Step 1: Remove the lamp by unscrewing

both screws and unplugging it.

Step 2: Install the lamp into the housing

(auto-aligning) and tighten screws.

Step 3: Check wavelength calibration

Step 4: Check lamp intensity (for future

reference)

Deuterium Lamp (1000 hours): 2140-0590

Deuterium Lamp (2000 hours): 2140-0820*

*includes RFID tag read by SL modules

Tungsten lamp G1103-60001

Sensitivity Data rate – Peak height

min 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

mAU

0

20

40

60

80

100 Data rate 80 Hz

Data rate 10 Hz

min 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

mAU

0

10

20

30

40

50

60

62 mAU

95 mAU

To

lua

mid

e

To

lue

ne

Peak height increases with

increasing data rate!

Wavelength Calibration test

Tip # 9: Care of Detector Flow Cells

Avoid the use of alkaline solutions with pH > 9.5 which can attack

quartz and impair optical performance.

Prevent crystallization of buffers or salts which will lead to blockage

and damage.

Aqueous solvents can allow algae growth. Don’t leave 100% water

standing in the flow cell. When leaving LC idle, pump a mobile phase

with at least 5-10% of organic solvent.

Observe the pressure limits of flow cells. Be careful when using

detectors in series or fraction collectors.

Summary - 1100/1200 LC Maintenance Overview

Solvent inlet Pump Autosampler Detector

Clean or Exchange:

Solvent inlet filter

Exchange:

PTFE frit

Pump seals

Outlet Ball Valve

AIV Cartridge

Wash seals

Clean:

Pistons

Support ring

Check:

Piston springs

Leak Sensor

Drain tube

Exchange:

*Needle

*Needle seat

Rotor seal

Check:

Leak Sensor

Drain tube

Exchange:

*Lamp

*Cell window

Clean:

Flow Cell

Check:

Leak sensor

Drain tube

* ... if necessary

Tests

Column

Compartment

Replace the rotor seal

(Column valve)

Check:

Leak sensor

Drain tube

Delete temporary files using the Disk Cleanup utility.

Use the Check Disk utility to scan and repair errors on the disk.

Defragment the hard drive. Use Virus detection software.

Have a back-up strategy for data, methods, sequences, custom UV-libraries, custom report templates, etc.

Make a copy of the bootptab file, Configuration Editor settings, and ChemStation license numbers.

Tip # 10 – Don’t forget the Data System Maintaining the ChemStation

Troubleshooting Key Concept

Divide and Conquer

Is the problem with the application method or the instrument?

Question

Inject a standard test mix of known behavior under

known conditions

Action

...............…HPLC Test Sample – Keep it simple:

Test sample: 2- 3 Analytes (chemically stable) in sufficient concentration

Test column: Record length, internal diameter, stationary phase, particle

diameter

Separation method: Solvent , flow rate, temperature, pressure, detection

parameters

Results: tR, peak width, resolution, peak area, peak height, baseline noise,

signal/noise ratio of analytes

Test Mix 01080-68704

C10H10O4

Dimethyl phthalate

C12H14O4

Diethyl phthalate

Biphenyl

ortho-terphenyl is C18H14

The Agilent Isocratic Test Mix 01080-68704

http://en.wikipedia.org/wiki/File:Dmp.png

http://en.wikipedia.org/wiki/File:Diethylphthalat.png

http://en.wikipedia.org/wiki/File:Bifenyl.svg

A typical chromatogram

min0.5 1 1.5 2 2.5 3 3.5 4 4.5

mAU

0

20

40

60

80

100

120

140

160

DAD1 A, Sig=254,4 Ref=360,100 (DEMO\LC_DEMO1\DEMO000003.D)

0.4

51 -

dim

ethy

phth

alat

e

0.5

91 -

die

thylph

thal

ate

1.0

69 -

bip

heny

l

1.9

76 -

o-te

rphe

nyl

Peaks elute to fast

Problem some peaks are lower

Where did that extra peak come from ?

40 percent less response from HPLC 1 than HPLC 2

HPLC 1

HPLC 2

9x times the height and area

Peak Area s varying retention times correct

Noisy Baseline

0.02 mAU

2.0 mAU

No Peaks

The Good

The Bad

Peaks elute to slow

The End – Thank You!

Agilent Tech Support: 800-227-9770

www.agilent.com/chem

1200 Series

HPLC and RRLC

6000 Series

LC/MS and

LC/MS/MS

tips and tricks of lc user maintenance...tips and tricks of lc user maintenance presented by greg...

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