Chromatography / Hints and tips


Many GC and LC problems can be avoided with routine preventive maintenance. If you are seeking the cause of the chromatography problem, go step by step. Never make more changes in your instrument at the same time, otherwise you will never find, what caused the problem.

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GC troubleshooting

GC troubleshooting

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LC troubleshooting

Signal to noise ration improvment in GC

GC chromatogramToday's laboratory needs are:

  • Lower detection and quantitation limits (LOD, LQD)
  • Stability improvment in GC and GC/MS systems
  • More inert and stable GC parts (columns, septa, vials, liners, ...)

Lower detection and quantitation limits can be achieved by:

  • Reducing the Noise
  • Increasing the signal

GC septa selection

Agilent GCs

Injector type Instrument Dimensions
Split-splitless 7890, 6890, 6850, 5890, 5880A 11 mm
Split-splitless 5880, 5700 9.5 / 10 mm
PTV 7890, 6890, 6850, 5890, 5880A 11 mm
On-column 7890, 6890, 6850, 5890 5 mm


Injector type Instrument Dimensions
Split-splitless Master, GC1000 12 mm
PTV Master, GC1000 12 mm

Perkin-Elmer GCs

Injector type Instrument Dimensions
Split-splitless Auto SYS, Auto SYS XL, 8000, 900, 990, Sigma 11 mm

Shimadzu GCs

Injector type Instrument Dimensions
Split-splitless, PTV 2010, 2014, 17A "plug-septa"

Varian GCs

Injector type Instrument Dimensions
Packed column   9.5 / 10 mm
1079, 1078   10 / 11 mm
1177   9 mm
1075 / 1077   11 mm

Thermo Scientific GCs

Injector type Instrument Dimensions
Split-splitless Trace, 8000, 8000 TOP 17 mm
PTV 8000 17 mm
Split-splitless Trace, GC9001 9.5 mm


Syringe Cleaning and Maintenance

Chromatography syringes are the finest quality precision fluid measuring devices available. With proper care and handling, syringes will provide unsurpassed performance in precision fluid measuring year after year. The life of your syringe is directly related to its cleanliness!

Some solvents, such as halogenated hydrocarbons, may attack and deteriorate the highly resistive adhesives (cements) used to affix needles and other terminations to Hamilton syringes, which may result in frozen plungers and plugged needles.


Cleaning Syringe Barrels

To clean Hamilton syringes, it is best to use solvents known to be effective in solvating the sample and preferably are non-alkaline, non-phosphate and non-detergent based. A biodegradable, non-phosphate, organic Cleaning Concentrate is available from Hamilton (ordering number 18311).

Rinse the syringe thoroughly after use with deionized water, acetone, or another solvent compatible with the sample. Allow the syringe to air dry. Avoid prolonged immersion of the syringe while cleaning.

MICROLITER™ Syringes (Series 600, 700, 800 and 900)
  • Rinse the syringe thoroughly with a solvent known to be effective in solvating the sample. Residual dissolved solids may result in frozen plungers and plugged needles.
  • To clean the plunger, remove it from the syringe barrel and gently wipe with a lint-free tissue. Reinsert the plunger into the barrel and pump deionized water, acetone or another solvent compatible with the sample through the needle and syringe. Allow syringe to air dry. When working with dissolved solids, storing the plunger outside of the syringe will reduce the possibility of frozen plunger.
GASTIGHT® Syringes (Series 1000, 1700 and 1800)
  • Rinse the syringe thoroughly with a solvent known to be effective in solvating the sample. Residual dissolve d solids may result in frozen plungers and plugged needles.
  • To clean the plunger, remove it from the syringe barrel and gently wipe with a lint-free tissue. Insert the plunger into the barrel and pump deionized water, acetone or another solvent compatible with the sample through the needle and syringe. Allow syringe to air dry. When working with dissolved solids, storing the plunger outside of the syringe will reduce the possibility of frozen plunger.
Syringe Storage

We recommend to store syringes in the original packaging. This with help to protect the syringe, and allows for easy identification. Remove product description label from the end of the box, and placing it to the outside packaging. This will make re-ordering the same syringe quick and easy.

ChromShell columns and solvents

There are several critical characteristics that must be taken into consideration when selecting the appropriate organic solvent to use in the mobile phase with ChromShell columns. Viscosity is one of the most important as high viscosity solvents may produce backpressures that are too high for the HPLC system used. Other important solvent characteristics include UV cutoff, cost and polarity index; where a solvent with a high UV cutoff will result in poor sensitivity with UV/Vis detection and use of high cost solvents will result in a poor laboratory that can't afford to buy new columns. Solvents with low polarity indices generally result in faster elution of organic compounds and are commonly used for column cleaning.


is arguably the best organic solvent as it results in the lowest system backpressure in water mixtures and also has a very low UV cutoff for better UV/Vis detection sensitivity. Although acetonitrile production is starting to increase with the turnaround of the economy, thus dropping cost, this still remains a major drawback of acetonitrile usage.


is another popular organic solvent as it is comparable in elution strength to acetonitrile, has a relatively low UV absorbance, and is significantly less expensive than acetonitrile. The major drawback of methanol, especially when used with small particle size HPLC columns, is that its use can result in backpressures that exceed many HPLC system limits.


is less commonly used as it has high UV absorbance, but can be used successfully if analytes absorb at higher UV wavelengths or if other detector types such as MS are used as it has similar elution properties to acetonitrile but is significantly less expensive.


is generally not recommended as it results in very high backpressures in water mixtures.

Iso-, n-propanol

have relatively strong elution strength and are most commonly used in column cleaning at low flow rates as they also results in high backpressures.


has similar elution strength to n-propanol but is less commonly used as it is much more expensive.

Glassware deactivation

Glassware deactivation with DMDCS

Dimethyldichlorsilane (DMDCS) reacts with active hydroxyl groups present on glass surface producing a deactivated layer. This ensures inert glassware used for sensitive compounds.


During the deactivation process, the reaction releases hydrogen chloride (HCl). So we strongly recommend to do this procedure in a fume hood.

  • Use 5% DMDCS solution in toluene. You can prepare this solution by diluting 20 ml of DMDCS with 400 ml toluene. Store the solution in an amber glass at room temperature.
  • Soak glassware in 5% DMDCS solution for 15 to 30 minutes.
  • Rinse glassware twice with toluene.
  • Soak glassware in methanol for 15 minutes.
  • Rinse glassware with methanol.
  • Dry glassware with high purity nitrogen (moisture and hydrocarbon free).

Setting linear velocity

The linear velocity is an important parameter in chromatography. It has an influence on chromatography resolution and therefore setting the dead volume is basic part of method development.

Linear velocity measurement in GC

To set a dead time, inject 2 µl of a non-retained gaseous substance, which is compatible with the detector. Take a gas-tight syringe and draw the headspace over neat compound. Accurately mark the injection starting time and peak elution time.

Recommended compounds for dead volume determination
Detector Compound
FID methane, propane, butane
ECD methylen chloride, air (at lower oven temperatures)
TCD methane, butane, air (at lower oven temperatures)
NPD acetonitrile, air (at lower oven temperatures)
MS propane, butane, argon, air (at lower oven temperatures)
PID acetylene, ethylene

Note: Some compounds may be slightly retained on thick-film phase, however, they will be reproducible for similar column types.

Handling chiral columns

Lux column use and care

Chiral columnShipping Solvent

n-Hexane/2-propanol (9:1, v/v)

Test Certificate

Each column is individually tested before shipment. A test certificate showing the separation parameters for trans-stilbene oxide is enclosed with each column.

Mobile Phase Compatibility

Lux columns can be used with normal phase (alkane/alcohol), reversed phase (aqueous methanol, aqueous acetonitrile or appropriate buffer/methanol or buffer/acetonitrile mixtures), as well as with pure polar organicsolvents (low molecular weight alcohols, acetonitrile or their mixtures).

Solvent Switching

An appropriate column washing procedure must be applied when changing from one mobile phase to another. The miscibility of the different mobile phase components must be carefully considered for this wash. To safely transfer a column from hexane to methanol (or acetonitrile) or from methanol (or acetonitrile) to hexane, use 100 % 2-propanol as transition solvent at a flow rate of 0.2-0.5 mL/min. Ten column volumes of 2-propanol (i.e. 25 mL for a 250 x 4.6 mm i.d. column or 15 mL for a 150 x 4.6 mm i.d. column) are sufficient for completely removing the old mobile phase. To safely transfer a column from normal phase to reversed phase conditions flush the column with 100 % 2-propanol at 0.2-0.5 mL/min for minimum ten column volumes. In addition, when the buffer salt additive of the RP mobile phase is insoluble in 2-propanol, flush the column briefly with water before switching to a buffered mobile phase. We recommend the use of dedicated Lux columns to reversed phase operation hence avoiding the need of converting columns used in normal phase elution mode to reversed phase or vice versa.

Use of Mobile Phase Modifiers

For basic samples or acidic chiral compounds, it may be necessary to use an appropriate mobile phase modifier in order to achieve chiral resolution and to insure proper peak shapes. Diethylamine, ethanolamine and butyl amine in the concentration range 0.1-0.5 % can be used with basic analytes, while trifluoroacetic or acetic acid (0.1-0.5 %; typically 0.1-0.2 %) with acidic analytes. Mixtures of basic and acidic mobile phase additives are acceptable (e.g. diethyl amine acetate or trifluoroacetate). Lux columns will deliver consistent results when operated with mobile phases containing additives at the concentration levels specified above. However, limited decrease in column efficiency may occur when a column is used in combination with these additives. Therefore, we advise to dedicate columns to mobile phases containing basic additives. Mobile Phase Restrictions Lux chiral stationary phases are prepared by coating silica with various polysaccharide derivatives. Therefore, any solvent dissolving the polysaccharide derivative (such as tetrahydrofurane, acetone, chlorinated hydrocarbons, ethylacetate, dimethylsulfoxide, dimethylformamide, N-methylformamide, etc.) must be avoided even in trace amounts (e.g. even as sample solvent).

Operating Backpressure

The mobile phase flow rate should be set such that the column backpressure stays below 300 bar (4300 psi). This maximum backpressure should not be exceeded for long periods of time.

Operating Temperatures

With standard mobile phases (such as alkane/alcohol) the column can be used in the temperature range 0-50 °C.

Column Storage

Column storage for a longer period of time is recommended in n-hexane/2-propanol (9:1, v/v). Columns used in reversed phase conditions should be first flushed with water (whenever a buffer salt was used as RP mobile phase additive) and then with methanol (or with methanol only when no salt was used). The column can be stored in methanol.

Extending Lifetime and Reconditioning

Phenomenex recommends the use of SecurityGuard™ guard cartridges to extend the lifetime of your column, especially with samples extracted from complex matrixes. Ideally, samples must be completely dissolved in the mobile phase or filtered through a syringe filter of approximately 0.45 μm porosity.

Hints and tips

LaboratoryThis site includes technical information, hints and tips for your decisions, and recommendation for selecting right chromatography accessories.

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