CAPILLARY GC INLETS. Dial 1-816-650-0741 for e-Seminar Audio SAMPLE INJECTION Goals Introduce sample into the column Reproducible No efficiency losses

CAPILLARY GC INLETS

Dial 1-816-650-0741 for e-Seminar Audio

SAMPLE INJECTIONGoals

• Introduce sample into the column

• Reproducible

• No efficiency losses

• Representative of sample


ShortConcentrated

LongDiffuse

Solute Bands

Same column, same chromatographic conditions

Influence of Injection Efficiency


FIRST, SOME BASIC DEFINITIONS:

• Backflash

• Discrimination

• Electronic pressure control (EPC)


BACKFLASHCause

• Vaporized sample expands 100 -1000 X

• Portions may leave the liner

• Occurs when vapor volume > liner volume


Sample expands to fill injector Some sample flows out of injectorHeavier portions condense on the cool areasNext injections or carrier gas dislodges condensed sample Then sample enters the column

BACKFLASH


10

20

30

40

50

60

70

80

90

50 100 150 200 250 300 350 400

Injection PortSet Point Temperature

350°C

35°COven

150°COven

300COven

Temperature in Gas Stream (°C)

Bottomof Septum

SyringeTip

Base ofInjection

Port

Temperature Profile of a Typical VaporizationInjector vs Oven Temperature


BACKFLASHProblems

• Loss of sample

• Baseline interferences

• “Ghost” peaks

• Tailing solvent front


BACKFLASHMinimizing

• Large volume liner

• Small injection volume

• Low expansion solvent

• Low injector temperature

• High carrier gas flow rates

• High head pressures


DISCRIMINATION

• Injected sample ¹ Sample into the column

• Due to compound volatility differences

• Higher volatility = More into the column


DISCRIMINATIONHydrocarbons at the Same Concentration

DB-1, 30 m x 0.32 mm ID, 0.25 um80-200°C at 10°/min, He at 30 cm/secSplit 1:100

2 4 6 8 10 12

C10

C11

C12C13


DISCRIMINATIONConsiderations

• Efficient heat transfer to injected sample

• Efficient mixing of vaporized sample with carrier gas

• Column position in inlet

• Inlet versus syringe discrimination

• Consistent conditions are important


ELECTRONIC PRESSURE CONTROLEPC

• Ability to program column head pressure

• Provides carrier gas flow or average linear velocity control during a run


CARRIER GAS PROPERTIES

• Compressible

• Viscosity increases with temperature

• Solute diffusion coefficients (Dm) increase with temperature


VELOCITY AND TEMPERATURE

• Average linear velocity decreases as column temperature increases

• Thus, average linear velocity changes during a temperature program


VELOCITY AND PRESSURE

• For a temperature program of 100-300°C

• Need to increase pressure by up to 7 times to maintain constant u


*Non-constant velocity or flow

EPC PRESSURE PROGRAMMINGIncrease Pressure During the GC Run

• Constant average linear velocity

• Constant flow

• Variable pressure program*


0

50

100

150

200

250

300

0 5 10 15 20 25 30 35

Time

Ove

n T

emp

erat

ure

0

5

10

15

20

25

30

35

40

Pre

ssu

re

Constant PressureConstant Pressure

Constant FlowConstant Flow

Pressure ProgramPressure Program

Oven TemperatureOven Temperature

EPC PRESSURE PROGRAMMINGTypes of Programs


EPC PRESSURE PROGRAMMINGBenefits

• Potential resolution improvements

• Reduce run times

• Lower elution temperatures

• Extend operating range for high temperature applications


EPC PRESSURE PROGRAMMINGCautions

• Pressure programming may improve, degrade or not affect resolution

• Dependent on column temperature program

• Accurate predictions are not possible


*Typically results in 10% run time decrease and 2-3% gain in resolution

EPC PRESSURE PROGRAMMINGStarting Points

• Try constant flow first*

• Then try higher final pressure and faster ramp rates


EPC PRESSURE PULSINGFor Splitless Injections

• High head pressure at the start of the GC run

• Followed by rapid decrease to desired head pressure


1. Minimizes sample expansion volume2. Rapid transfer of analytes into the column

EPC PRESSURE PULSINGBenefits

• Reduce loss of more volatile analytes1

• Reduce analyte decomposition in the injector2

• Better reproducibility with large volume injections


Especially for earlier eluting peaks

EPC PRESSURE PULSINGCautions

• Peak broadening may occur

• A retention gap is recommended


EPC PRESSURE PULSINGStarting Points

• Limit initial pressure to 2-3 times normal column head pressure

• Start with 0.25 min pulse time

• Pulse times >1.0 min usually result in distorted peaks


EPC MAINTENANCELeaks

• Inability to reach set points

• Pressure/retention time fluctuations

• More sensitive to leaks than mechanical systems


EPC MAINTENANCEMost Common Problems

• Need to occasionally zero EPC channels

• Electronic valves are more prone to plugging and sticking

• Contamination by high MW compounds at high concentrations


OTHER INLET CONSIDERATIONS

• Silylation

• Glass or fused silica wool

• Cleaning and reusing liners


GLASS WOOLConsiderations

• Always use deactivated (silylated) wool

• Borosilicate or quartz material?


GLASS WOOLLiner Packing Recommendations

• Amount, size and placement must be consistent for consistent results

• Can be broken upon installation into the liner, exposing active sites

• Liner deactivation with glass wool plug in place is ideal


GLASS WOOLPlacement in Liner

• Near top of liner:

• Wipes syringe needle of sample

• Can improve injector precision

• Helps to prevent backflash

• Near bottom of liner:

• Helps in volatilization of high MW components

• Increases mixing


LINER DEACTIVATION

• Prior to deactivation, surface must be cleaned with an acid leach step:

• Place liner in clean test tube

• Cover liner with 1N HCl or HNO3 solution

• Soak for at least 8 hours (overnight is preferred)

• If acid solution is highly discolored, replace with clean solution and

continue to soak until no color change is noted

• Do not soak liners for longer than 24 hours

• Rinse with deionized water followed by methanol

• Dry the liner at 100-150°C. Do not exceed 150°C.


LINER DEACTIVATION

• Solution Silylation Procedure

• Place liner in screw cap test tube

• Cover liner with 10% TMCS or DMCS in toluene

• Tightly seal with Teflon-lined cap

• Allow to stand for at least 8 hours

• Remove from solution and thoroughly rinse with toluene, then methanol

• Dry the liner at 75-100°C

• NOTE: Several liners can be done in one test tube, but rotate the tube several times to ensure that all surfaces are

exposed to the solution.


COMMON INJECTION MODES

• Vaporization Injection Modes

• Cold Injection Modes

• Large Volume Injection Modes

COLD INJECTION MODES



• Thermally unstable solutes

• Low volatility solutes

• Dilute samples (on-column)



• PTV and Cold On-column injection

• The sample is injected into a controlled temperature environment

• The solute bands need refocusing prior to beginning the separation


COLD ON-COLUMN INJECTION

• Clean samples

• Thermally labile solutes

• High boiling solutes

• Refocusing required


J&W COLD ON-COLUMN INJECTOR DESIGN


GROB COLD ON-COLUMN INJECTOR DESIGN


Cool TowerNeedle Guide

Duckbill Valve(Isolation Valve)

Septum purge out

Cryogenic cooling(optional)

Carrier in

Column

Heater block

Spring

Insert

GC

Septum

For Autoinjection

COLD ON-COLUMN INJECTION PORT


SECONDARY COOLING

• Some means used to cool a small section at the front of the column:

Forced air

Liquid N2 or CO2

Column extraction from oven


J&W COLD ON-COLUMN INJECTOR DESIGNSample Refocusing


GROB COLD ON-COLUMN INJECTOR DESIGNSample Refocusing


Cool TowerNeedle Guide

Duckbill Valve(Isolation Valve)

Septum purge out

Cryogenic cooling(optional)

Carrier in

Column

Heater block

Spring

Insert

GC

Septum

For Autoinjection

COLD ON-COLUMN INJECTION PORTSample Refocusing


SECONDARY COOLING Benefits

• Warmer initial oven temperatures

• Shorter cool-down between analyses

• Shorter analysis times


SECONDARY COOLING Considerations

• Potential peak shape problems

Split peaks

Band broadening

• Retention gap should be used


*see Knaus, Fulleman & Turner, HRC 4(1981)643

At Injection (To)

Needle At Injection (To)

Carrier Gas

Carrier Gas

Solvent Vapors

Column

Column

Temperature ProfileHigh volatility solute

Low volatility solute

“SECONDARY COOLING” CAN CAUSE SPLIT PEAKS*!

Retention gap is usually a necessity

SECONDARY COOLINGPeak Splitting


THE RETENTION GAP

• A 1 to 2 mtr length of uncoated but deactivated fused silica tubing, attached to the front of the column with a press-fit connector, is usually adequate. The diameter is usually equal to or greater than that of the column.


COLD ON-COLUMN INJECTION REFOCUSING


Volatiles Focused by Solvent

High Boilers Focused by Stationary Phase

Solvent Evaporating from the rear of the flooded zone

Solvent Evaporates

Flooded zone forms in the retention gap

Retention gap

Stationary Phase


SYRINGE TECHNIQUE: ON-COLUMN INJECTIONPlunger-in-needle type of syringe may be preferred.

Manufacturer’s recommendations should be consulted. Fewer problems are encountered with small injections. Outside of needle must be dry.. avoid sample contact between the needle and the column; this would result in a “smeared” band. Retention gap useful and often critical…see “On Column Injection in Capillary Gas Chromatography”, K. Grob, Huethig Publishing, 1988


PTV INJECTORS

• Lower thermal mass

• Rapid heating and cooling

• Lower internal volume

• Packing options

• Split vent timing


Split LineCapillary Column

Insert(vaporization Chamber)

Glass Wool / Packing

Carrier Gas

Heating Coil

Seal

Septum PurgeSeptum

Cooling Gas

Cooling Gas

PTV INJECTION PORT


Capillary Column

Carrier Gas

Heating Coil

Cooling DeviceSplit Line

Injection Needle


PTV INJECTION MODES

• Cold on-column

• Cold split

• Cold splitless


COLD ON-COLUMN Using PTV Injector

• Same rules that exist for non-PTV applications still apply:

Secondary cooling can be used

Sample refocusing still required


COLD SPLIT VS COLD SPLITLESSWhich To Use?

• The same general concepts that apply to standard vaporization techniques also apply to Cold PTV injection techniques:

Split: General use and sample screening

Splitless: Trace level samples


PTV COLD INJECTION TECHNIQUESBenefits Over Standard Vaporization Techniques • Reduced inlet discrimination

• Reduced decomposition of labile analytes

• Less risk of backflash contamination*

• Larger injection volumes

• *More prevalent with Splitless injections


PTV COLD INJECTION CONDITIONSDuring Injection

PTV inlet PTV inlet ColdCold ColdColdModes Modes Split Split SplitlessSplitless

Liner temperature <<Solvent BP <<Solvent BP

Purge/Split flow OFF OFF

Delay before NONE NONEinlet heating


PTV inlet PTV inlet ColdCold ColdColdModes Modes Split Split SplitlessSplitless

Liner temp program Maximum rate To max temp in<80 sec

Column temperature Solute dependent <Solvent BP

Purge/Split flow Split-ratio dependent Off until finaltemp reached

PTV COLD INJECTION CONDITIONSDuring Inlet Heating


n-Paraffin standard showing distillation range from C6 to C110 on DB-HT Sim Dist

Column: DB-HT Sim Dist5 m x 0.53 mm I.D., 0.15 µm

Carrier: Helium at 18 mL/min, measured at 35°COven: - 30 - 430°C at 10°/minInjector: OPTICTM PTV

55 - 450°C at 2°/sec0.5 µL of about 2% n-Parraffins in CS2

Detector: FID, 450°CNitrogen makeup gas at 15 mL/min

Time(minutes)0 5 10 15 20 25 30 35 40 45 50

6

7

8

9

10

16

1112

70

50

40

9080

60

14

18

20 24 2830

32

110

HIGH TEMP SIM DIST ANALYSISn-Paraffins


LARGE VOLUME INJECTION


LARGE VOLUME INJECTIONBenefits

• Enhanced detection and recognition

• Quicker more accurate sample preparation

• Better coupling to automated systems


LARGE VOLUME INJECTION

• PTV

• Cool On-Column


ON-COLUMN LARGE VOLUME INJECTION

• Longer retention gap

• Solvent removal

• Very large volumes


On-columninjector Detector

Solvent vaporexit

restriction

Retention Gap

RetainingPrecolumn

AnalyticalColumn




PTV LARGE VOLUME INJECTION

• Solvent elimination split/splitless

• Rapid at once

• Controlled injection rate


PTV SOLVENT ELIMINATION INJECTION Conditions During Injection

PTV Inlet PTV Inlet Solvent Elimination Solvent Elimination Solvent EliminationSolvent EliminationModes Modes Split Split SplitlessSplitless

Liner Temperature <Solvent BP <Solvent BP (vapor-pressure dependent)

Purge/Split Flow 100-1000 mL/min 100-1000 mL/min

Delay before 5-30 sec 5-30 secinlet heating


PTV Inlet PTV Inlet Solvent Elimination Solvent Elimination Solvent EliminationSolvent EliminationModes Modes Split Split SplitlessSplitless

Liner temp program Maximum rate To max temp in <80 sec

Column temperature Solute dependent <Solvent BP

Purge/Split flow split-ratio dependent Off until final inlettemp reached

PTV SOLVENT ELIMINATION INJECTION Conditions During Inlet Heating



Larger inner diameter PTV liners can retain large volumes

Approximate volumes retained (hexane)

Liners packed with glass wool plug (3 cm)C.A. Cramers, H.G. Janssen, H.G.J. Mol, U.A.T. Brinkman,

J. High Resolution Chromatography

Liner Diameter Liner Diameter VolumeVolume

1.2 mm 20 µL

2.2 mm 65 µL

3.4 mm 150 µL

RAPID “AT ONCE” INJECTIONMaximum Sample Volumes



LARGE VOLUME INJECTION CONSIDERATIONS

• Use only high purity solvents (GC/MS grade)

• Store small volumes of solvent (£ 500 mL)

• Optimize inlet conditions to prevent solvent breakthrough during injection (PTV)


ON-COLUMN LARGE VOLUME INJECTION

• Most inert

• Less thermal stress

• Volatile analysis

• Very large volume

• Ruggedness (“clean samples”)

• Speed control of injection


PTV LARGE VOLUME INJECTION

• Dirty samples

• Easy optimization (“at once” and controlled injection)

• Packing selectivity

• Labile analysis


Sample

Volatile Range Organics

Purge and Trapor

Static Headspace

Concentrated

ThermallyStable

PTV Splitor

On-Column

High BoilingComponents

No

No

Concentration

ThermallyStable

Yes

High BoilingPoint

Splitless

PTV Splitlessor

On-Column

Dilute

No Yes

NoYes

NoYes

Split


GO FORTH AND INJECT!!!


J&W Scientific Technical Support

800-227-9770 (phone: US & Canada)*

302-993-5304 (phone)*

* Select option 4, then

option 2.916-608-1964 (fax)www.agilent.com/chem


Wrap-up E-Seminar Questions

Thank you for attending Agilent e-Seminars.

Our e-Seminar schedule is expanding every

week. Please check our website frequently at:

www.agilent.com/chem

Documents

CAPILLARY GC INLETS. Dial 1-816-650-0741 for e-Seminar Audio SAMPLE INJECTION Goals Introduce sample into the column Reproducible No efficiency losses