column phase selection guide troubleshooting tools ... - Phenomenex

Polar 

TROUBLESHOOTING TOOLS 

• Flow meter 

• Leak detector 

• New syringe 

• Column performance test sample 

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. 

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. 

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 

• Methane or other non-retained compound 

• Reference column 

• New septa, ferrules and injector liners 

• Instrument manuals 

• 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. 

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 

Correct 

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. 

In order to minimize the downtime of theinstrument, 

columns can be conditioned overnight at the maximum 

isothermal temperature. 

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. 

COLUMN DIMENSION SELECTION GUIDE 

RESOLUTION 

(R) 

SAMPLE CAPACITY 

(SC) 

RETENTION TIME 

(t r 

) 

Incorrect 

Length (L) ID (r) Phase Thickness (d f ) 

R ~ √ — L 

To double resolution, 

quadruple the length 

SC ~ √ — L 

Longer columns have 

slightly better capacity 

t r ~ L 

Longer columns require 

longer analysis times 

CHECKING FOR LEAKS 

Use a thermoconductivity detector to check for leaks. 

It is highly sensitive to H 2 

, He, and N 2 

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. 

R ~ r 

Resolution decreases with 

increased diameter 

SC ~ r 2 

Capacity is exponential 

as diameter increases 

t r ~ r 

Smaller diameter columns 

allow faster analysis times 

R ~ 1 

√ — d f 

Resolution decreases with increased thickness 

SC ~ d f 

Sample capacity increases with thicker films 

t r ~ d f 

Thicker films require longer analysis times 

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. 

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 

NON-RETAINED PEAK TIMES AND MARKERS 

Type Marker Compound 

Methane with FID/TCD: 

Detector 

Calculate linear velocity by injecting 

ECD 

Linear Velocity (u) = L/t o 

25-100 μL of 1 % methane in N 2 gas blend. 

Measure the retention time of the 

FID 

NPD 

Methane, Butane 1 

Acetonitrile 2, 4 

PID ELCD 

Vinyl chloride 

methane peak and calculate the following: 

TCD, MS 

COLUMN PHASE SELECTION GUIDE 

Polarity Scale 

5 

8 

9 

13 

18 

19 

24 

52 

57 

58 

Temperature MS 

Phase 

Highlight Composition Polarity Range* Certiftied 

ZB-1 Non-polar phase suited for true boiling point separations 100 % Dimethylpolysiloxane Nonpolar -60 to 360/370 °C 3 

ZB-1ms Extremely low bleed column for non-polar compounds 100 % Dimethylpolysiloxane Nonpolar -60 to 360/370 °C 3 

ZB-1HT Inferno High temperature stability (430 °C) for non-polar 100 % Dimethylpolysiloxane Nonpolar -60 to 400/430 °C 3 

compounds 

ZB-5 Versatile low polarity column for general lab use 5 %-Phenyl 

95 %-Dimethylpolysiloxane 

Nonpolar -60 to 360/370 °C 3 

ZB-5ms 

ZB-5MSi 

ZB-5HT Inferno 

ZB-XLB 

Arylene phase for enhanced resolution of PAHs and 

multi-ring aromatic compounds 

Very inert for good peak shape of active compounds, such 

as acids and bases 

High temperature stability (430 °C) to separate high 

molecular weight compounds and eliminate carry overs 

Low polarity si-arylene column with bleed and 

sensitivity levels designed for MS detectors 

5 %-Phenyl Arylene 


5 %-Phenyl 


Nonpolar -60 to 325/350 °C 3 

Nonpolar -60 to 360/370 °C 3 

5 %-Phenyl 


Nonpolar -60 to 400/430 °C 3 

Proprietary Intermediate 30 to 340/360 ºC 3 

ZB-XLB-HT High temperature anlaysis of mid-polar compounds Proprietary Intermediate -60 to 400 ºC 3 

Inferno 

ZB-624 

ZB-35 

Optimized for separating volatile organic compounds 

(VOCs) 

Intermediate polarity column for high molecular weight 

analysis 

6 %-Cyanopropylphenyl 


35 %-Phenyl 


ZB-35HT Inferno High temperature analysis of mid-polar compounds 35 %-Phenyl 


ZB-1701 

ZB-1701P 

ZB-50 

ZB-WAXPLUS 

ZB-WAX 

Unique selectivity providing an alternate selectivity to 

phenyl phases with similar polarity 

Specially tested to ensure good response for DDT and 

endrin 

High polarity column capable of high-temperature bakeout 

to remove contaminants 

100 % aqueous stable with high retention of alcohols and 

other chlorinated solvents 

Bonded, solvent rinsable phase excellent for separating 

polar complex mixtures 

ZB-FFAP Provides better peak shape for underivatized acids Nitroterephthalic Acid 

Modified Polyethylene Glycol 

ZB- MultiResidue 

MR-1 & MR-2 

Novel phase designed for pesticides, herbicides, 

and insecticides 

Intermediate -20 to 260 °C 

Intermediate 50 to 340/360 °C 3 

Intermediate -60 to 400 ºC 3 

14 %-Cyanopropylphenyl 


Polar -20 to 280/300 °C 

14 %-Cyanopropylphenyl Polar -20 to 280/300 °C 


50 %-Phenyl 

Polar 40 to 320/340 °C 3 


Polyethylene Glycol Polar 20 to 250/260 °C 

Polyethylene Glycol Polar 40 to 250/260 °C 3 

Polar 40 to 250/260 °C 

Proprietary Intermediate -60 to 320/340 °C 3 

ZB-Bioethanol Fast analysis of bioethanol fuel Proprietary -60 to 340/360 ºC 3 

ZB-Drug-1 Rapid and improved separations for drugs of abuse Proprietary -60 to 320/340 ºC 3 

ZB-BAC1 & 

ZB-BAC2 

More accurate blood alcohol analysis, especially for post 

mortem samples 

Proprietary -20 to 260/280 ºC 3 

* See individual phases for detailed specifications and limitations. 

Methylene chloride 2, 3 , Dichlorodifluoromethane 

Methane, Butane 1 , air 

1. From a disposable lighter 

2. Place 1-2 drops in an autosampler vial and tightly cap. Shake and inject 1-2 μL from 

the headspace of the vial. Do not inject any liquid. 

3. Use a column temperature above 55 ºC. 

4. Use a column temperature above 95 ºC. 

RECOMMENDED NON-RETAINED RETENTION 

TIMES 

Length (m) H 2 (sec) He (sec) N 2 (sec) 

Non-Polar 

15 38 75 150 

30 75 150 300 

60 150 300 600 

Tailing peak indicates 

improper installation 

Symmetrical peak indicates 

proper installation 

h (u) Curves for Different Gases 

Zebron is a registered trademark of Phenomenex, Inc. Inferno, Engineered Self 

Cross-linking, Arylene Matrix Technology, and MultiResidue are trademarks of 

Phenomenex, Inc. Vespel is a registered trademark of E.I. du Pont de Nemours 

and Co. © 2010 Phenomenex, Inc. All rights reserved. 

Australia 

t: 02-9428-6444 

f: 02-9428-6445 

auinfo@phenomenex.com 

Canada 

t: (800) 543-3681 

f: (310) 328-7768 

info@phenomenex.com 

France 

t: 01 30 09 21 10 

f: 01 30 09 21 11 

franceinfo@phenomenex.com 

Italy 

t: 051 6327511 

f: 051 6327555 

italiainfo@phenomenex.com 

The Netherlands 

t: 030-2418700 

f: 030-2383749 

nlinfo@phenomenex.com 

Puerto Rico 

t: (800) 541-HPLC 

f: (310) 328-7768 


www.phenomenex.com 

Phenomenex products are available worldwide. For the distributor in your country, 

contact Phenomenex USA, International Department at international@phenomenex.com 

Austria 

t: 01-319-1301 

f: 01-319-1300 

anfrage@phenomenex.com 

Belgium 

t: +31 (0)30-2418700 

f: +31 (0)30-2383749 

beinfo@phenomenex.com 

Denmark 

t: 4824 8048 

f: 4810 6265 

nordicinfo@phenomenex.com 

Finland 

t: (09)4789 0063 

f: +45 4810 6265 


Germany 

t: 06021-58830-0 

f: 06021-58830-11 


Ireland 

t: 01 247 5405 

f: +44 1625-501796 

eireinfo@phenomenex.com 

Luxembourg 

t: +31 (0)30-2418700 

f: +31 (0)30-2383749 


Mexico 

t: (55) 5018 3791 

f: (310) 328-7768 

tecnicomx@phenomenex.com 

New Zealand 

t: 09-4780951 

f: 09-4780952 

nzinfo@phenomenex.com 

Norway 

t: 81 00 20 05 

f: +45 4810 6265 


United Kingdom 

t: 01625-501367 

f: 01625-501796 

ukinfo@phenomenex.com 

All other countries: 

Corporate Office USA 

t: (310) 212-0555 

f: (310) 328-7768 

info@phenomenex.com

PEAKS 

NO PEAKS 

GHOST PEAKS 

TAILING PEAKS 

BROAD SOLVENT FRONTS 

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 ............................................................ 


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 ......................................... 


• Contaminated or active injector liner or column ............ 

• Dead volume due to poorly installed liner or column ..... 

• Ragged column end ...................................................... 

• A bad match between the polarities of stationary ......... 

phase and the solvent 

• A cold region in the sample flow path ........................... 

• Debris in the liner or column ......................................... 

• Injection takes too long ................................................. 

• Split ratio is too low ....................................................... 

• Overloading the inlet .................................................... 

• Some types of compounds such as alcoholic ............... 

amines and carboxylic acids tend to tail 


• Bad column installation ................................................. 

• Injector leak .................................................................. 

• Injection volume too large ............................................. 

• Injection temperature too low ........................................ 

• Split ratio is too low ...................................................... 

• Column temperature too low ......................................... 

• Initial column temperature too high ................................ 

for splitless injection 

• Purge time too long (splitless injection) ......................... 

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. 


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. 


Clean or replace injector liner. Don’t use glass wool in the liner. Solvent rinse or replace 

the column. 

Confirm by injecting inert peak (methane). If it tails, column is not properly installed. 

Reinstall liner and column as necessary. 

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. 

Change the stationary phase. Usually polar analytes tail on non-polar columns, 

or dirty columns. 

Remove any cold zones in the flow path. 

Clean or replace the liner. Cut 10 cm off the end of the column and reinstall it. 

Improve injection technique. 

Increase split ratio to at least 20:1. 

Decrease the sample volume or dilute sample. 

Try a more polar column. Make a derivative of the sample. 


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. 

Increase split ratio. 

Increase column temperature. Use a lower boiling point solvent. 

Decrease the initial column temperature. Use a less volatile solvent so the initial column temperature 

is at least 10 °C below the boiling point of the solvent. Use a shorter purge activation time. 

Use a shorter purge activation time. 

BASELINE 

POSSIBLE CAUSES 

FOR BASELINE INSTABILITY 

SYSTEM 

• Leaks, O 2 influx 

• Column bleed 

• Septum bleed 

• Contaminated gases 

• Unequilibrated 

• Dirty detector 

• Dirty inlet 

• Improper flow rates 

• Pneumatic temperature change 

REFERENCE 

PARAMETERS UNIT SYMBOLS 

Retention Time of 

an Unretained Solute 

Retention Time, 

Measured from the Start 

s t o 

Adjusted Retention Time s t ’ R = t R - t o 

Peak Width 

(Base) 

Peak Width 

(Half Height) 

s 

s 

s 

t R 

W 

W1/2 

t o 

COLUMN 

• Bleed contamination 

• Liquid leak detector 

contamination 

Capacity Factor 

– 

(Retention Factor) 

k = t ’ R 

t o 

k 

Selectivity Factor – 2 t ’ 

a = = 

R2 

k 1 t’ R1 

t’ 

Resolution – R2 

- 

R s = 2 

t, R1 

( W 1 + W 2 

) 

Number of Theoretical Plates – N = 5.54 

t R t 

(W1/2) 2 R 

= 16( W ) 2 

t’ 

Number of Effective Plates – R t’ 

N eff = 5.54 (W1/2) 2 R 

= 16 ( W) 2 

Column Length cm L 

Height Equivalent of a 

Theoretical Plate (Plate Height) 

cm H = 

Effective Plate Height cm H eff = 

Linear Velocity of the 

Mobile Phase 

Pressure Conversions 

cm s -1 U = 

L 

N 

L 

N eff 

L 

t o 

1 bar = 100 kPa 

1 atm = 101.3 kPa 

1 psi = 6.9 kPa 

SAMPLE 

• Carry over 

• Depolymerization agents 

(HCl, KOH, etc.) 

HOW TO 

DECREASE 

PEAK WIDTH 

1. Use a More 

Efficient Column 

• Packed - 

smaller particles, packed 

more tightly 

• Capillary - 

smaller ID, thinner film 

2. Optimize Method 

Parameters 

• 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 manufacturer’s 

recommended 

Installation Instructions 

• Eliminate any leaks 

• Optimize detector flows 

FRONTING PEAKS 

SPLIT PEAKS 


• Column overloading ....................................................... 


• 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. 


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. 


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. 

NON-REPRODUCIBLE RETENTION TIMES 


System 

• Leaks ............................................................. 

• Erratic flow controller ...................................... 

• Unstable oven temperature .............................. 

• Pneumatic temperature change ...................... 

• Line pressure change ...................................... 

• Injection technique .......................................... 

Column 

• Polarity changing from contamination .............. 

• Adsorption ....................................................... 

Sample 

• Concentration solute/stationary ....................... 

phase insolubility 


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. 

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. 

Increase final temperature in program with hold time. Remove 50-100 cm 

from injector side of column. 

Use retention gap. Change column phase. 

Contact Phenomenex to get your 

FREE COPY of our GC Troubleshooting Guide 

PO52200710_I 

Australia 

t: 02-9428-6444 

f: 02-9428-6445 

auinfo@phenomenex.com 

Canada 

t: (800) 543-3681 

f: (310) 328-7768 


France 

t: 01 30 09 21 10 

f: 01 30 09 21 11 

franceinfo@phenomenex.com 

Italy 

t: 051 6327511 

f: 051 6327555 

italiainfo@phenomenex.com 

The Netherlands 

t: 030-2418700 

f: 030-2383749 


Puerto Rico 

t: (800) 541-HPLC 

f: (310) 328-7768 


www.phenomenex.com 

Phenomenex products are available worldwide. For the distributor in your country, 

contact Phenomenex USA, International Department at international@phenomenex.com 

Austria 

t: 01-319-1301 

f: 01-319-1300 


Belgium 

t: +31 (0)30-2418700 

f: +31 (0)30-2383749 

beinfo@phenomenex.com 

Denmark 

t: 4824 8048 

f: 4810 6265 


Finland 

t: (09)4789 0063 

f: +45 4810 6265 


Germany 

t: 06021-58830-0 

f: 06021-58830-11 


Ireland 

t: 01 247 5405 

f: +44 1625-501796 

eireinfo@phenomenex.com 

Luxembourg 

t: +31 (0)30-2418700 

f: +31 (0)30-2383749 


Mexico 

t: (55) 5018 3791 

f: (310) 328-7768 

tecnicomx@phenomenex.com 

New Zealand 

t: 09-4780951 

f: 09-4780952 

nzinfo@phenomenex.com 

Norway 

t: 81 00 20 05 

f: +45 4810 6265 


United Kingdom 

t: 01625-501367 

f: 01625-501796 

ukinfo@phenomenex.com 

All other countries: 

Corporate Office USA 

t: (310) 212-0555 

f: (310) 328-7768 

info@phenomenex.com

column phase selection guide troubleshooting tools ... - Phenomenex

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