TROUBLESHOOTING TOOLS

Flow meter Leak detector New syringe Column performance test sample

Methane or other non-retained compound Reference column New septa, ferrules and injector liners Instrument manuals

UNRETAINED VOLUME PEAK SHAPE TESTING

If the peak is broad and/or tailing, check the following: Improper column positioning/insertion into inlet or detector Gross contamination of the splitter sleeve Chipped or cracked splitter sleeve Improper sweeping of sample at column end by makeup gas Damaged or crushed column end
Tailing peak indicates improper installation Symmetrical peak indicates proper installation

PRE-INSTALLATION CHECK LIST

Replace oxygen, moisture and hydrocarbon traps as necessary. Check gas cylinder pressures to ensure that an adequate supply of carrier, make-up and fuel gases are available. Carrier gases should be of the highest purity.  Note: It is critical that oxygen and water be removed from the carrier gas by the appropriate use of filters and adsorbents. Ensure that the injection port is clean and free of sample residues, septum, or capillary debris. Check and replace as necessary critical injector components such as seals, liners, and septa. Check and replace detector seals as necessary. Carefully inspect your column for damage or breakage.

NON-RETAINED PEAK TIMES AND MARKERS

Methane with FID/TCD: Calculate linear velocity by injecting 25-100 L of 1 % methane in N2 gas blend. Measure the retention time of the methane peak and calculate the following: Linear Velocity (u) = L/t o
Detector Type ECD FID NPD PID ELCD TCD, MS
1. 2. 3. 4.

CRITICAL COLUMN INSTALLATION STEPS

INJECTOR INSTALLATION:
Recommendation: GC columns do not have a specific directional flow when received from the manufacturer. Upon initial use of your new Zebron column, Phenomenex recommends the practice of dedicating one specific end of the column for injector installation only. This is particularly important when dealing with active/caustic or contaminating compounds. If these compounds are routinely injected onto the column, degradation of the phase will occur - leading to higher bleed. A typical first step to remedying (removing) this bleed would be to trim 10 cm from the front (injector) end of the column and keep trimming this inlet end of the column as necessary. Trying to remedy any bleed issues by trimming the column may not work if both ends have been interchangeably installed into the inlet. 1. Place a capillary nut and ferrule on the injector end of the GC column, allowing a section of column to protrude. Trim one to two centimeters from the protruding end to remove ferrule contamination that may have entered the column. Inspect the cut with a magnifier to ensure that a smooth, clean, square-cut edge has been made - recut if necessary. 2. Carefully hang the column in the GC oven, being cautious not to scratch or damage the polyimide coating on the capillary tubing. Rotate the column to avoid sharp bends of the capillary column and any contact of the column with oven surfaces. 3. Insert the column into the injector exactly the correct distance specified in the instrument manual. Tighten the ferrule nut finger-tight then 1/2 turn with a wrench. If the column can still be moved, tighten another 1/4 turn until the column is secure. 4. Adjust the carrier gas to obtain the flow rate listed on the test chromatogram. pointing in the correct direction. Inspect the cut with a magnifier and ensure that the cut is square and smooth. Recut if needed. 2. Insert the outlet end of the column into the detector exactly the distance prescribed in the instrument manual. Distances will vary between detectors. Tighten the ferrule nut finger-tight then 1/2 turn with a wrench. If the column can still be moved, tighten another 1/4 turn until the column is secure. 3. Inspect the column connections for leaks using an electronic leak detector. Leaks at the inlet end may introduce oxygen to the column that will result in increased column bleed and damage to the column phase. Proper & Improperly Cut Capillary

Marker Compound 2, 3 Methylene chloride , Dichlorodifluoromethane 1 Methane, Butane Acetonitrile2, 4 Vinyl chloride Methane, Butane1, air

From a disposable lighter Place 1-2 drops in an autosampler vial and tightly cap. Shake and inject 1-2 L from the headspace of the vial. Do not inject any liquid. Use a column temperature above 55 C. Use a column temperature above 95 C.

RECOMMENDED NON-RETAINED RETENTION TIMES

Length (m) 15 30 60 H2 (sec) 38 75 150 He (sec) 75 150 300 N2 (sec) 150 300 600 h (u) Curves for Different Gases

Correct

Incorrect

COLUMN PHASE SELECTION GUIDE

Polarity Scale Phase
ZB-1

COLUMN CONDITIONING:
1. Allow sufficient time for the carrier gas to flow through the column to purge any oxygen that may be in the system. 2. Raise the temperature of the column to the maximum isothermal operating temperature that is listed on the individual Zebron GC Column Test Report. Maintain this temperature until a constant baseline is achieved. Conditioning times will depend on the phase identity and thickness, with thicker films taking longer to stabilize. If necessary in order to minimize the downtime of the instrument, columns can be conditioned overnight at the maximum isothermal temperature.

ZB-1ms

Applications Composition Polarity Essential oils, gases (refinery), hydrocarbons, MTBE, 100 % Dimethylpolysiloxane Nonpolar natural gas odorants, oxygenates and GROs, semi-volatiles, simulated distillation, solvent impurities, sulfur compounds (light) Amines, acids, diesel fuel, drugs of abuse, flavors & 100 % Dimethylpolysiloxane Nonpolar fragrances, pesticides, polychlorinated biphenyls (EPA 1668) High boiling petroleum products, simulated distillation methods, long-chained hydrocarbons, high molecular weight waxes, polymers/plastics, diesel fuel, motor oils Alkaloids, dioxins, drugs, essential oils/flavors, FAMEs, halo-hydrocarbons, PCBs/aroclors, pesticides/herbicides, phenols, residual solvents, semi-volatiles, solvent impurities Acids, alkaloids, amines, dioxins, drugs, essential oils/flavors, EPA Methods, FAMEs, halo-hydrocarbons, PCBs/aroclors, pesticides/herbicides, phenols, polyaromatic hydrocarbons (PAH), residual solvents, semi-volatiles, solvent impurities High boiling petroleum products, simulated distillation methods, long-chained hydrocarbons, high molecular weight waxes, triglycerides, diesel fuel, motor oils, surfactants, polymers/plastics Specifically designed for the separation of volatile organic compounds (VOCs). Widely used phase to separate residual solvents in industrial or pharmaceutical products (OVIs). Excellent for US EPA Methods 501.3, 502.2, 503.1, 524.2, 601, 602, 624, 8010, 8015, 8020, 8240, 8260, 8021 Aroclors, amines, nitrogen containing pesticides, organochlorine pesticides, organophosphorous pesticides, pharmaceuticals, semi-volatiles, EPA Method 508, 608, 8081, 8141, 8151 Alcohols, amines, aromatic hydrocarbons, PAHs, PCBs, pesticides/herbicides, phenols, steroids, tranquilizers 100 % Dimethylpolysiloxane Nonpolar

Temperature MS Range* Certiftied -60 to 360/370 C

-60 to 360/370 C -60 to 400/430 C

Non-Polar

Non-Polar

ZB-1HT Inferno

ZB-5

5 %-Phenyl 95 %-Dimethylpolysiloxane 5 %-Phenyl Arylene 95 %-Dimethylpolysiloxane

Nonpolar

-60 to 360/370 C

ZB-5ms

Nonpolar

-60 to 325/350 C

DETECTOR INSTALLATION:
Note: For users with sensitive detectors such as MS and ECD, column conditioning steps should be performed before installing the column to prevent contamination and frequent maintenance of the detector. 1. Place the column nut and ferrule past the end of the column and cut a centimeter or two off the end of the column. Be sure that the ferrule is the right size and

INSTALLATION TESTING:
1. Inject a detectable unretained sample, such as methane for an FID, to determine dead volume time and linear gas velocity at the desired column temperature. Adjust gas pressure for optimal flow depending on carrier gas selection. 2. The non-retained peak must have ideal peak shape or installation is faulty and needs to be redone.
Polar

ZB-5HT Inferno

5 %-Phenyl 95 %-Dimethylpolysiloxane

Nonpolar

-60 to 400/350 C

13 18 19

ZB-624

6 %-Cyanopropylphenyl 94 %-Dimethylpolysiloxane

Intermediate -20 to 260 C

ZB-35

35 %-Phenyl 65 %-Dimethylpolysiloxane

Low-Mid Polar

50 to 340/360 C

COLUMN DIMENSION SELECTION GUIDE

Length (L) RESOLUTION (R)

R~L

ID (r)

Phase Thickness (d f)

ZB-1701

14 %-Cyanopropylphenyl 86 %-Dimethylpolysiloxane 14 %-Cyanopropylphenyl 86 %-Dimethylpolysiloxane 50 %-Phenyl 50 %-Dimethylpolysiloxane Polyethylene Glycol

Polar

-20 to 280/300 C

To double resolution, quadruple the length SAMPLE CAPACITY (SC)

SC ~ L tr ~ L

Resolution decreases with increased diameter

~ r

R ~
df
Resolution decreases with increased thickness

SC ~ r tr ~ r

24 52
Polar

Longer columns have slightly better capacity Longer columns require longer analysis times

Capacity is exponential as diameter increases Smaller diameter columns allow faster analysis times

SC ~ df
Sample capacity increases with thicker films

RETENTION TIME (t r )

t r ~ df
Thicker films require longer analysis times

57
58

ZB-1701P Organochlorine pesticides, nitrogen containing pesticides, organophosphorous pesticides, aroclors ZB-50 Antidepressants, aroclors, cholesterols, drugs of abuse (especially basic), glycols, pesticides/herbicides, steroids, triglycerides, EPA Methods 508, 608, 8081, 8141, 8151 ZB-WAX Alcoholic beverages, alcohols, aldehydes, aromatics, essential oils, flavors/fragrances, glycols, pharmaceuticals PLUS OVIs, solvents, styrene, xylene isomers ZB-WAX Alcohols, aldehydes, aromatics, essential oils, flavors & fragrances, glycols, OVIs, pharmaceuticals, solvents, styrene, xylenes isomers, basic compounds ZB-FFAP Acrylates, alcohols, aldehydes, free fatty acids, ketones, organic acids, phenols, volatile free acids

Polar

-20 to 280/300 C

Intermediate 40 to 320/340 C

Polar

20 to 250/260 C

Polyethylene Glycol

Polar

40 to 250/260 C

Nitroterephthalic Acid Polar Modified Polyethylene Glycol

40 to 250/260 C

CHECKING FOR LEAKS

Use a thermoconductivity detector to check for leaks. It is highly sensitive to H2, He, and N2 and will not contaminate the instrument or column. Liquid leak indicators are not recommended for capillary columns. There is the risk of drawing the liquid into the column or fittings and contaminating the system.

NOTE: If Vespel ferrules are being used, leakage can occur after the initial heating phase due to ferrule deformation. Be sure that the fitting is re-tightened after this initial heating phase, then carefully check all corrections for leaks.

MultiResidue Columns Organochlorine pesticides, nitrogen containing pesticides, Proprietary (MR)-1 insecticides, haloacetic acids, organophosphorous pesticides, herbicides, aroclors/PCBs, multi-pesticide residue methods MultiResidue Columns Organochlorine pesticides, nitrogen containing pesticides, Proprietary (MR)-2 insecticides, haloacetic acids, organophosphorous pesticides, herbicides, aroclors/PCBs, multi-pesticide residue methods

Intermediate -60 to 320/340 C

Intermediate -60 to 320/340 C

* See individual phases for detailed specifications and limitations.

www.phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or email: international@phenomenex.com.
mail:

Australia PO Box 4084 Lane Cove, NSW 2066 Australia 02-9428-6444 02-9428-6445 info@ phenomenex.com.au

Austria Zeppelinstr. 5 63741 Aschaffenburg Germany 01-319-1301 01-319-1300 anfrage@ phenomenex.com

Canada 411 Madrid Ave. Torrance, CA 90501-1430 USA (800) 543-3681 (310) 328-7768 info@ phenomenex.com

Denmark Gydevang 39-41 3450 Allerd Denmark 4824 8048 4810 6265 dkinfo@ phenomenex.com

France Parc des Grillons, Bat.3 60 route de Sartrouville 78232 Le Pecq Cedex France 01 30 09 21 10 01 30 09 21 11 franceinfo@ phenomenex.com

Germany Zeppelinstr. 5 63741 Aschaffenburg Germany 06021-58830-0 06021-58830-11 anfrage@ phenomenex.com

Ireland Queens Avenue, Hurds eld Ind. Est., Maccles eld, Cheshire SK10 2BN, UK 01 247 5405 +44 1625-501796 eireinfo@ phenomenex.com

Italy Via Emilia, 51/C 40011 Anzola dellEmilia (BO) Italy 051 736176 051 735302 italiainfo@ phenomenex.com

New Zealand P O Box 31-601 Milford 0741 North Shore City New Zealand 09-4780951 09-4780952 info@ phenomenex.co.nz

Puerto Rico 273 Sierra Morena, Suite #104 San Juan, Puerto Rico 00926 (800) 541-HPLC (310) 328-7768 info@ phenomenex.com

United Kingdom Queens Avenue, Hurds eld Ind. Est., Maccles eld, Cheshire SK10 2BN, UK 01625-501367 01625-501796 ukinfo@ phenomenex.com

USA 411 Madrid Ave. Torrance, CA 90501-1430 USA (310) 212-0555 (310) 328-7768 info@ phenomenex.com

tel.: fax: email:

BASELINE
POSSIBLE CAUSES FOR BASELINE INSTABILITY

PEAKS
NO PEAKS
Possible Cause: System Clogged syringe ............................................................ Leaks ............................................................................ No carrier gas ............................................................... Detector OFF or not lit ................................................... Wrong injection port ...................................................... Clogged inlet sleeve ...................................................... Column Broken column .............................................................. Plugged column ............................................................ Suggested Remedy: Clean or replace syringe. Check injector for leaks. Make sure column is properly installed in detector. Turn on carrier gas. Ignite or turn on detector. Reduce sample size or gas flows if solvent blew out detector. Verify correct injection port. Replace inlet liner. Inspect column and verify flow at column outlet. Cut off 5-10 cm of column ends and reinstall column. Verify flow at column outlet.

SYSTEM Leaks, O2 influx Column bleed Septum bleed Contaminated gases Unequilibrated

Dirty detector Dirty inlet Improper flow rates Pneumatic temperature change

COLUMN Bleed contamination Liquid leak detector contamination

SAMPLE Carry over Depolymerization agents (HCl, KOH, etc.)

REFERENCE
PARAMETERS UNIT s s s s s cm cm cm SYMBOLS to tR t R = t R - to W Retention Time of an Unretained Solute Retention Time, Measured from the Start Adjusted Retention Time Peak Width (Base) Peak Width (Half Height) Capacity Factor (Retention Factor) Selectivity Factor Resolution Number of Theoretical Plates Number of Effective Plates Column Length Height Equivalent of a Theoretical Plate (Plate Height) Effective Plate Height Linear Velocity of the Mobile Phase Pressure Conversions

GHOST PEAKS
Possible Cause: System Septum bleed ................................................................ Carry over ..................................................................... Dirty inlet ...................................................................... Contaminated gas ......................................................... Outgassing from traps .................................................. Contaminated gas lines ................................................. Column Sample contaminated ................................................... Sample Contaminated sample ................................................... Contaminated flush solvent ........................................... Possible sample degradation ......................................... Suggested Remedy: Replace septum with high-temperature, low-bleed septum. Increase final temperature and hold. Rinse syringe better. Replace inlet liner. Replace filters, scrubbers, or service purifiers. Use higher purity gasses. Replace traps. Replace or clean gas lines. Cut 50-100 cm from injector side of column. Perform an extended conditioning step. Solvent rinse column. Use glass wool in liner or decrease injection temperature, or replace column. Remake sample with higher purity/fresh solvents and clean glassware. Replace syringe flush solvent with fresh/pure solvent. Make new sample. Store samples using proper procedures. Reduce introduction of catalysts or reactive analytes in sample. Store samples in opaque or dark containers.

HOW TO DECREASE PEAK WIDTH

1. Use a More Efficient Column
Packed - smaller particles, packed more tightly Capillary - smaller ID, thinner film

W k= a= t R to k2 k1 = t R2 t R1 t R2 - t
,

TAILING PEAKS
Possible Cause: Suggested Remedy: Contaminated or active injector liner or column ............ Clean or replace injector liner. Dont use glass wool in the liner. Solvent rinse or replace the column. Dead volume due to poorly installed liner or column ..... Confirm by injecting inert peak (methane). If it tails, column is not properly installed. Reinstall liner and column as necessary. Ragged column end ...................................................... Score the tubing lightly with a sapphire scribe or a ceramic scoring wafer before breaking it. Examine the end. If the break is not clean and the end square, cut the column again. Point the end down while breaking it, and reinstall the column while installing a nut and ferrule. This will prevent fragments from entering the column. A bad match between the polarities of stationary ......... Change the stationary phase. Usually polar analytes tail on non-polar columns, phase and the solvent or dirty columns. A cold region in the sample flow path ........................... Remove any cold zones in the flow path. Debris in the liner or column ......................................... Clean or replace the liner. Cut 10 cm off the end of the column and reinstall it. Injection takes too long ................................................. Improve injection technique. Split ratio is too low ....................................................... Increase split ratio to at least 20:1. Overloading the inlet .................................................... Decrease the sample volume or dilute sample. Some types of compounds such as alcoholic ............... Try a more polar column. Make a derivative of the sample. amines and carboxylic acids tend to tail

2. Optimize Method Parameters

R1

Rs = 2

tR 2 N = 5.54 W = 16

t R 2 t R 2 Neff = 5.54 W = 16 W L H= Heff = L N L N eff L to

( ) ( ) ( ) ( ) ( )
W1 + W2

tR 2 W

See van Deemter Plots for optimal flow rates of carrier gases Optimize temperature profiles

3. Reduce Sample Size

To avoid column overloading

4. Reduce Dead Volume in System

Follow manufacturers recommended Installation Instructions Eliminate any leaks Optimize detector flows

BROAD SOLVENT FRONTS

Suggested Remedy: Reinstall column. Find and fix leak. Decrease sample size or dilute 1:10. Increase injection temperature so the entire sample is vaporized instantly. An injection temperature higher than the temperature limit of the column will not damage the column. Split ratio is too low ...................................................... Increase split ratio. Column temperature too low ......................................... Increase column temperature. Use a lower boiling point solvent. Initial column temperature too high ................................ Decrease the initial column temperature. Use a less volatile solvent so the initial column temper for splitless injection ature is at least 10 C below the boiling point of the solvent. Use a shorter purge activation time. Purge time too long (splitless injection) ......................... Use a shorter purge activation time.  Possible Cause: Bad column installation ................................................. Injector leak .................................................................. Injection volume too large ............................................. Injection temperature too low ........................................

cm s-1 U =

1 bar = 100 kPa 1 atm = 101.3 kPa 1 psi = 6.9 kPa

FRONTING PEAKS
Possible Cause: Column overloading ....................................................... Suggested Remedy: Reduce the injection volume (increasing sensitivity, if necessary), increase split ratio, or use a column with greater capacity. Columns with larger diameters or thicker stationary phase coatings generally have larger sample capacities; however, resolution may be reduced.

NON-REPRODUCIBLE RETENTION TIMES

Possible Cause: System Leaks ............................................................. Erratic flow controller ...................................... Unstable oven temperature .............................. Pneumatic temperature change ...................... Line pressure change ...................................... Injection technique .......................................... Column Polarity changing from contamination .............. Suggested Remedy: Check injector for leaks. Make sure column is properly installed in detector. Verify flows. Fix/replace flow controller if necessary. Calibrate oven. Possibly replace thermocouple. Redirect oven exhaust. Regulate room temperature. Install dual stage regulator. Standardize a reproducible injection technique.

SPLIT PEAKS
Possible Cause: Poor (jerky or erratic) injections ..................................... Bad column installation ................................................. Fluctuations in column temperature .............................. Mixed sample solvent for splitless ................................ or on-column injections When using injection techniques that ............................ require solvent effect refocusing such as splitless injection, the solvent must form a compact, continuous flooded zone in the column. If the solvent does not wet the stationary phase (column lining) sufficiently, as might be the case for methanol used with a nonpolar stationary phase, the solvent flooded zone may be several meters long and not of uniform thickness. This will result in broad and distorted peaks because the solutes will not be refocused into a narrow band near the beginning of the column. Suggested Remedy: Use smooth, steady plunger depression. Use autosampler. Reinstall column. Repair temperature control system. Calibrate GC oven. Use single solvent. Installing a retention gap (5 meters of uncoated but deactivated column) ahead of the chromatographic column may reduce or eliminate this problem.

Cut 50-100 cm from injector side of column; perform an extended conditioning step. Solvent rinse column. Use glass wool in liner or decrease injection temperature; or replace column. Adsorption ....................................................... Increase final temperature in program with hold time. Remove 50-100 cm from injector side of column. Sample Concentration solute/stationary ....................... Use retention gap. Change column phase. phase insolubility
Vespel is a registered trademark of E.I. du Pont de Nemours and Co. Arylene Matrix Technology, Engineered Self Cross-linking, Inferno, MultiResidue, and Zebron are trademarks of Phenomenex, Inc. 2007 Phenomenex Inc. All rights reserved.

www.phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or email: international@phenomenex.com.
mail:

Australia PO Box 4084 Lane Cove, NSW 2066 Australia 02-9428-6444 02-9428-6445 info@ phenomenex.com.au

Austria Zeppelinstr. 5 63741 Aschaffenburg Germany 01-319-1301 01-319-1300 anfrage@ phenomenex.com

Canada 411 Madrid Ave. Torrance, CA 90501-1430 USA (800) 543-3681 (310) 328-7768 info@ phenomenex.com

Denmark Gydevang 39-41 3450 Allerd Denmark 4824 8048 4810 6265 dkinfo@ phenomenex.com

France Parc des Grillons, Bat.3 60 route de Sartrouville 78232 Le Pecq Cedex France 01 30 09 21 10 01 30 09 21 11 franceinfo@ phenomenex.com

Germany Zeppelinstr. 5 63741 Aschaffenburg Germany 06021-58830-0 06021-58830-11 anfrage@ phenomenex.com

Ireland Queens Avenue, Hurds eld Ind. Est., Maccles eld, Cheshire SK10 2BN, UK 01 247 5405 +44 1625-501796 eireinfo@ phenomenex.com

Italy Via Emilia, 51/C 40011 Anzola dellEmilia (BO) Italy 051 736176 051 735302 italiainfo@ phenomenex.com

New Zealand P O Box 31-601 Milford 0741 North Shore City New Zealand 09-4780951 09-4780952 info@ phenomenex.co.nz

Puerto Rico 273 Sierra Morena, Suite #104 San Juan, Puerto Rico 00926 (800) 541-HPLC (310) 328-7768 info@ phenomenex.com

United Kingdom Queens Avenue, Hurds eld Ind. Est., Maccles eld, Cheshire SK10 2BN, UK 01625-501367 01625-501796 ukinfo@ phenomenex.com

USA 411 Madrid Ave. Torrance, CA 90501-1430 USA (310) 212-0555 (310) 328-7768 info@ phenomenex.com

tel.: fax: email:

5220_L

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