LC Consumables Tips and Tricks - labfarmasi.com · 7 January 25, 2019 Title Confidentiality label Regulatory statement ... Avoid exposure to direct sunlight (useAluminum foil, shield

LC ConsumablesTips and Tricks

Chromatography Workflow

FY 2019

Solution

Suryadi

Sales Chemical Ultra Standard & Consumable-Chromatography Sipplies

Agilent Technologies

[email protected]

Maret 01, 2019

mailto:[email protected]

Chromatography Workflow

SampleCollection

Analysis

SamplePreparation

Detection

Separation

2 January 25, 2019 Title Confidentiality label Regulatory statement (if applicable)

Mobile Phase Reservoir


Care of Mobile Phase

Particulates in the LC solvents or microbial growth in the solvent bottle may plug thesolvent 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 amber glass bottles.

• Consider adding 5-10% organic to the aqueous mobile phase to inhibit microbial growth.


Solvent and Buffer Filtration

As internal diameter of connecting capillaries, particle in the column and pore size of columninlet frit are getting smallermust.

and smaller, filtration of mobile phase is becoming an absolute

HPLC use 0.45 m filter

UHPLC use 0.2 m filter

p/n: 3150-0577


PN Description

5191-4339 Filter membrane, PTFE, 47 mm, 0.20 µm, 100/pk

5191-4336 Filter membrane, PTFE, 47 mm, 0.45 µm, 100/pk

5191-4341 Filter membrane, nylon 47 mm, 0.20 µm, 100/pk

5191-4338 Filter membrane, nylon 47 mm, 0.45 µm, 100/pk

5191-4340 Filter membrane, cellulose 47 mm, 0.2 µm, 100/pk

3150-0576 Filter membrane, regenerated cellulose, 47 mm, 0.45 μm, 100/pk

Solvent Inlet Filter

1.

2.

3.

Remove the solvent inlet filter.

Keep a clean one on hand to compare

Replace the glass filter (or soakconc. H2NO3 for 1 hour.

Flush well with water

in

4.

5. Reinstall the solvent inlet filter

p/n: 5062-8517 p/n: 5041-2168 p/n: 01018-60028


InfinityLab Solvent Bottles

• Agilent’s new solvent bottles have been

redesigned for easier use and safer

handling, lowering the risk ofthe lab.

accidents in

• The new solvent bottles feature an

ergonomic design

and ergonomically

on both sides of

with a slimmer profileshaped gripping zones

the bottle, promoting

easier and safer usage.


Solvent Bottles

• Borosilicate type 1 ClassA2 glass reservoirs.

• Wash glassware separately from other containers.

• Clean reservoirs – dishwasher, no detergent.

(If more aggressive cleaning is required,

sonicate with 10% formic or nitric acid, then water,

then methanol or acetonitrile, repeat two more time)

• Final rinse with DI water.

• Put in 400 degree muffle furnace for 6 hours.


ProblemsRetention Time Shift

Key Factor Affecting pH and Concentration of Buffer

Evaporation


InfinityLab Stay Safe CapWasteMobile Phase and


Stay Safe Cap for Mobile Phase

The safety caps seal the solvent containers so that harmfulvapors do not escape into the laboratory.

The integrated exhaust valve maintains a constant

pressure in the solvent container and allows proper solvent

flow to the HPLC system.

The valve membrane opens when the

HPLC pump is operating and allows air

flow into the bottle, preventing a partial

vacuum inside the solvent container.

to

When the pump stops, the valve membrane

closes and solvent vapors are blocked

from escaping into the laboratory. Venting

valve should be replaced every 6 months

in order to flawless operation.


Open bottle (A) Standard cap (B) Agilent InfinityLab Closed cap (D)

Stay Safe cap (C)

5991-7755EN


A B C D


Acetonitrile MSDS


Methanol MSDS


Stay Safe Cap for Waste


Pump


Routine Maintenance Procedure

•

•

•

•

•

Replace

Replace

Replace

the

the

the

seal and pistons

PTFE frit

cartridge in active inlet valve

Clean

Clean

the outlet ball valve

or replace solvent inlet filter

p/n: 01018-22707


Autosampler


Injection Valve

Standard Autosampler G1329A

54

32

1

Injection valve

1.2.

3.

4.

5.

Stator screws

Stator head

Rotor seal

Isolation seal

Valve body

0100-18490100-1853 0101-1416

0100-1853: Rotor seal (Vespel)0100-1849: Rotor seal (Tefzel),

0101-1416: Rotor seal (PEEK)


Needle and Needle SeatStandard Autosampler G1329A

Needle seat

i.d. 0.17 mm: p/n G1313-87101

i.d. 0.12 mm: p/n G1313-871-3

Needle: p/n G1313-87201


Autosampler Vials


A-Line Vials


VialsSepta

& SeptaChemical Compatibility

5990-9022EN

29

Re-washed Glass Surface

scratched, abraded

and plaques

Re-washed Glass Surface

scratched, abraded

and plaques

To Reuse: Physical Deformations On The Glass Surface

New Glass Surface New Glass Surface

X100

X500

30

smooth and free of

surface scratches

and abrasions

smooth and free of

surface scratches

and abrasions

Amber GlassClear Glass

LC Columns Compartment


Problems!!!

• Peak shape•

•

Peak separation

Peak tailing

• Column blockage• High back pressure

• Column selection• Is it the right column for my application?


• Delays peak elution time.

• Broadens peaks reducing

efficiency and resolution.

Problem

Effect of Extra-column Volumes

• Decreases peak height and thus S/N.Resolution 0.961mAU

400

Peakwidth 0.037 min300

200


0

0.5 1 1.5 2 2.5 min

Resolution 1.902mAU300

250


150

100Peakwidth 0.018 min

50

0

0.5 1 1.5 2 2.5 min


HPLC Instrument Configuration

Design differences between LC instrumentation

– such as power range, delay volume, mixing

behavior, temperature control, extra column

volume and detector cellthe results of analysis.

design – all effect to

Delay volume

gradient mixing behavior

pressure x flow rate

Delay volumeextra column volume

Injection volume

Temperature profileextra column volume

Data rateextra column volume

path-length


Del

ayv

olu

me

Extr

a c

olu

mn

volu

me

Resolution

Retention Time

Sensitivity

Retention Time

Resolution

Retention Time

Resolution

Sensitivity

Retention Time

Resolution

FittingConventional LC


Limitations


Infinity A-Line Connector

InfinityLab Quick Connect UHPLC columnfittings for truly dead-volume-free fluidic connections

Finger tight up to•

•

Tool-free connection up to 1300 bar

Spring loaded design for easy and truly zero-dead volume connection

600 and 1300 bar

• Removabletypes

and reusable for all column


Zero-dead Volume

Agilent column with InfinityLab Quick Connect

Agilent column with InfinityLab Quick Turn

Too short screw

Competitor column with Swagelog

Dead volume

Competitor column with InfinityLabQuick Connect


Ease of Use

1. Screw the fitting (blue wheel) with lever in

“open” position onto the column – or column

onto the fitting

2. Stop when finger-tight and turn the lever

3. Finished… in a seconds


Finger tight does not mean as far as your finger can go

→ Stop as soon as you can feel the first resistance

Excessive force will damage the fitting!

ressureDetermining The Cause and Correcting High Back

Check pressure with/without column - many pressuredue to blockages in the system or guard column.

problems are

•

•

Remove column - pressure still high?

Remove guard - pressure still high?

If column pressure is high:

• Wash column

Eliminate column contamination and plugged packing

High molecular weight/adsorbed compounds

Precipitate from sample or buffer

•

•

•

• Change frit – Clear plugged frit PREVENT THIS!


etter Choice

s

el Regulatory statement (if applicable)

The TricksPrevent Technique—The B

• Use column protection

In-line filters

Guard columns

•

•

•

•

Filter samples

Filter buffered mobile phase

•

•

Sample clean-up (i.e. SPE)

Appropriate column flushing

43 January 25, 2019 Title Confidentiality lab

Use Appropriate Guard Column

Guard cartridges should be changed when there is a big increase in backpressure


Detectors


Detector in LC

• UV-Visible detector

• Diode array detector

• Fluorescence detector

• Refractive index detector

• Evaporative light scattering detector

• Electrical conductivity detector

• Electrochemical detector

• Mass spectrometer


Certified Lamp

By providing higher sensitivity, Agilent lamps can extend

detection capabilities and improve qualification at trace

levels – for more than 2,000 hours of use


Caring for your UV detector flow cell

• Many flow cells have quartz windows or fused silicacomponents, so avoid using alkaline solutions with

pH greater than 9.5, which can attack these components

and impair optical performance.

• To prevent crystallization inside the flow cell, always flushbuffers or salt solutions, especially at high pHs.

it with water after running

• When your LC is unused overnight, make sure your flow cell contains at least 10%organic mobile phase to prevent algae growth.

• Some modern flow cells use coated or uncoated fused silica capillaries. You canadversely affect coated capillaries by turning on the UV lamp without solvent flow, so

read the manufacturer ’s recommendations carefully.

48

Flushing The Flow Cell

If your system was shut down unexpectedly during an unattended sequencerun, it is possible that the sample is still trapped in your flow cell.

If the system was stopped overnight with salt buffer, it is important to start the

system flush with a lower flow rate and watch the system pressure. A warm-

water flush using the 60°C setting on your column compartment is an

effective way to clean your UV flow cell.

49



Habits of aHealthy

GC

Title

1Confidentiality label

Regulatory statement (if applicable)

w your GC and the respective consumables:Kno

Where can the issue be?

There are 7 basic areas from where the problem may arise:

1.

2.

3.

4.

5.

6.

7.

Injector

Gas Flow/Leaks

GC Column

Detector

Electronics

Gas Supply / Purity of Gases / Filters

Sample Storage - Vials and Closures

But of course it can always be a COMBINATION. A healthy GC can ELIMINATEsome of these problems before they begin to surface.

For example, Peak Tailing

Injector or Column is “Active”

- Reversible adsorption of active compounds like the ones with -OH, -NH, -SH

Flow path problem

-

-

Dead volume, obstruction, poor installation, leaks or

Severe column contamination.

Miscellaneous

- overloading of PLOT columns, co-elution, polarity mismatch between phase, solute or solvent, and some compounds always tail.

*Tip = Inject a light hydrocarbon, should not tail unless there is aflow path problem.

It all begins with “ Maintenance”It is not

always clear when I need to maintain

theinstrument

Sometimes Iforget to do

maintenanceWhen?

Is it time for maintenance? I’m not sure which task I

need toperform to correct or prevent a problem

I’d like some suggestions on a typical maintenance protocol and

best practices

What?

I needresources to improve my

maintenanceskills

I don’t feel comfortable

doing maintenance

How?

5 5 September 2018 Agilent Technologies

How often do I need to do complete routine maintenance?

More OftenLess Often


Recommended Maintenance Tasks


GC Maintenance Decision Tree


Column and Liner Contamination

GC Column Maintenance

1. Bake out the column forthan 2 hours.

no more

2. Cut off around 15-30cm of theinlet end of the column.

3. Cut off more column (repeat asnecessary)

Why Do You need to do this? Example of Column Contamination

Example of Column Contamination after cutting the column

Inlet MaintenanceWhy and when is it needed?• Every analysis means wear and

inlet partstear on

••

Every injection introduces contaminationHigh temperature stress of GC aggravateswear••

•

•

How many samples?What is the sample type?How critical are the results?Do you have a regular standard procedure,SOP for maintenance?

Inlet Maintenance Schedule

Part Many samples Less samples

heavy matrix clean samples

Septum Daily Weekly

Liner Daily Monthly

Gold seal/washer Weekly Monthly

O-ring With liner With liner

Ferrule With column With trimming/exchange

trimming

Split vent trap 3 months Yearly

What are some potential Causes of Split Peaks?

INJECTOR (poor sample introduction)- Injecting the sample twice or injection too slow(manual) or in needle

- Mixed sample solvent (polarity difference)

VOLATILITY- High boilers dropping out on Cold Spots- Transfer line temperature- Unions or fittings not tracking with column temp.

INJECTOR (activity)- Breakdown (not really a split peak, 2 peaks)- Sample degradation in injector

Don’t forget to change your split vent trap


Don’t forget to change your split vent trap


Addressing Endrin/DDT Breakdown


Addressing Endrin/DDT Breakdown


Agilent Inert Flow Path (IFP) Solution Focus

Deactivated Inlet Weldments,

Shell and Transfer LinesDeactivated – FPD, TCD,NPD/FID Jets

Ultra Inert Inlet Liner

UltiMetal Plus treatedFlexible Metal Ferrules

UltiMetal Tubing

Ultra Inert Press-FitUltiMetal Plus treatedCapillary Flow

Technology Devices

Ultra Inert GC ColumnUltra Inert Gold Seal

Gas Clean Gas Purifier

http://intranet.chem.agilent.com/Workspaces/Images1/Entitled Partner/082865/Shared Images/7890B_5977A_7693A_Tray_Front.tif

http://intranet.chem.agilent.com/Workspaces/Images1/Entitled Partner/082865/Shared Images/7890B_5977A_7693A_Tray_Front.tif

Why Ultra Inert?

Regulatory bodies drive for lower detection limits forincreasingly active and complex samples

Active flow paths causes

• Unreproducible performance

• Waste time, resources and hinder productivity

• Reduce capability of expensive equipment

• Result in sample reruns or reporting an incorrect result

These problems can all be avoided.column selection and maintenance

Let’s start with

Ultra Inert ColumnsPeak shape on DB-5ms UI

4-picoline TMP 1,2-pentanediol

App Note 5989-8665EN

Agilent J&W DB-5ms Ultra Inert

30m x 0.25mm x 0.25µm

5% phenyl Competitor

30m x 0.25mm x 0.25µm

http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-8665EN.pdf

Ultra Inert Inlet Liners

Ultra Inert deactivated inlet liners (Proprietary vapor deposition process) provides higherresponse for sensitive compounds.

Productivity:Touchless packaging with pre-installed o-ring:

quick & easy hassle free installation

Issue: Peak Response Variations

Injector or Column is active/contaminated----

Irreversible adsorption of active compoundsDecomposition of the sample

(-OH, -NH, -SH)

Temperature change or injector discrimination in the inletEvaporation of the sample

Contamination from Liquid Soap on fingers duringcolumn installation

Column:

Carrier:

Injector:

Detector:

Oven:

DB-5ms, 30m x 0.25mm, 0.25um

H2, 60 cm/sec, constant flow

split 1:20, 250CFID, 320C, N2 makeup gas

40C for 0.75 min, 40-325C at 20C/min,

325C for 30 min

Red: Liquid Soap

Blue: system blank

.

Ultra Inert Gold PlatedSeal

o Liner rests on the gold

seal

o The slit allows for the split vent

Cross slit for high split vents (>200ml/min)

o Gold surface important to seat on the

liner

Ultra Inert Gold Seals, Advantages

100 ppb STD

x103

500 ppb STD

x103

8

Ultra Inert Gold SealOmethoateAcephate

14Acephate Omethoate

00

x103x103

8

Siltek SS

1Acephate Omethoate4

OmethoateAcephate

00

x103 x103

8

Standard Gold Seal

1 Acephate Omethoate4

Acephate Omethoate

00

4 4.5 5 5.5 6 6.5 7 7.5 4 4.5 5 5.5 6 6.5 7 7.5

Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min)

Inert Pathway vs Standard PathwayDrugs

of Abuse

0.25ng on column

App Note 5991-1859EN

Inert Pathway:o Ultra Inert GC/MS column

o Ultra Inert Gold Seal

o Ultra Inert liner

o UltiMetal Plus inlet weldment

o UltiMetal Plus Flex. Ferrules

Do you see a lot of this in yourlab?

Bonus Peaks or Ghost Peaks

Contamination in the INJECTOR

••

Carry-over from a backflash or previous sampleSeptum bleed

COLUMN or… Carrier Gas

• Bad tank of gas or traps have expired

NoisyBaseline

FLOW- Contaminated gas- Incorrect detector settings

DETECTOR- Air leak - ECD, TCD- Electronics malfunction

COLUMN- Bleeding if at high temperatures- In detector flame (poor installation)

Non-indicating Gastraps

Safeguarding the Inert GC Flow Path:Gas Purification with Gas Clean CarrierGas

Optimizes your GC/MS performance

• Single combination Filter, optimized to remove

oxygen, moisture and hydrocarbons from

GC/MS

carrier gas

• Increased sensitivity with more accurate data

• Prevents column damage, lower replacement frequency

• Reduces instrument down-time

filter

Clean Gas Filters

SAVINGS!

And now, after lookinghuman

at chromatographic health and safety, here aresome practical health and safety issues

51 9/5/2018 Agilent Confidential

GC & GC-MS - Split Vent Trap

Did you know about the external split venttrap?

Universal/External

SplitVent Trap

Features:

• Trap analytes in

contamination

sample effluent to prevent

• When testing potentially hazardous samples,

the split vent flow should be vented to an

exhaust hood

• Easy to change cartridge design

• Economical insurance against costly

damage

Ultra Scientific

A Guideline for Standard

> Buffer and Reagent

> Pesticide, PAH, and Lipid Standard

> Inorganic Standard

TitleConfidentiality labelRegulatory statement (if applicable)52 January 25, 2019

About Ultra Scientific-Agilent

COA(Certificate of Analysis)

Buffers and Reagents

Conductivity Solutions

Pure Water – Titrants & Test Solutions

Inorganic Chemical Standard Ultra

Single Element Inorganic Standards

Agilent ISO Guide 34 certified reference materials (CRM)

Multi-Element Inorganic Standards

ICP-OES Calibration Standards

Pesticide, PAH, and Lipid Standards

Standar agilent, tersedia dalam Ampul, Vial, danBotol dapat digunakan untuk penggunaan langsungatau untuk penyimpanan jangka panjang. Untukhasil terbaik, standar analitik harus digunakansegera setelah dibuka, dan tidak perlu ditransferuntuk digunakan nanti. Standar Harus disimpandalam botol CERTAN adalah paket inovatif palingaman.Setiap komponen dalam standar referensi sudahdianalisis sebelumnya, dan pelarutnya memilikikualitas terbaik.Standar dikirimkan dengan sertifikat yangmenunjukkan kemurnian senyawa yang tersediauntuk setiap standar. Standar solusi disiapkansecara gravimetri dengan presisi ± 0,5%, dandikirimkan dengan sertifikat yang menunjukkankonsentrasi pestisida dalam standar.

Polynuclear Aromatic Hydrocarbon (PAH) Standards PAH adalah polutan persisten yang tersebar luas. Beberapamemiliki ikatan yang beracun atau karsinogenik. Semuabahan diproduksi di bawah sistem mutu Agilent ISO 9001.Sertifikat yang menunjukkan kemurnian senyawa tersedia untuksetiap standar.

Lipid Standards

Lipid Kits

Lipid Kits

Thank

You

Any Questions

LC & GC Consumable or CrossLabChemical Ultra Standard

[email protected] 1973 4416 Call or WA


mailto:[email protected]

Documents

LC Consumables Tips and Tricks - labfarmasi.com · 7 January 25, 2019 Title Confidentiality label Regulatory statement ... Avoid exposure to direct sunlight (useAluminum foil, shield