Split/Splitless Inlet Cleaning Procedure

6890 Split/Splitless Inlets

The Split/Splitless inlet will become active (degrading certain types of compounds) over time. In some cases, just changing the liner and the gold seal will not remedy this problem. In these cases a more vigorous cleaning procedure must be implemented. The following procedure has been developed to help in this process.

1. Cool the inlet. This is best accomplished by reducing the inlet temperature to 40C.

2. After the inlet has cooled sufficiently ( at least 70C), turn the inlet flow off.

3. If an autosampler is in place, remove tower, tray and top cover.

4. Remove the weldment assembly that covers the GC liner. Remove the liner completely from the GC. Notice a flow line that sits below the weldment, this is the split vent flow line. This should have a 7/16” swagelok fitting, loosen the fitting and remove the split vent flow line.

5. Loosen and remove the GC column nut from the inlet. Place a septa over the injection port end of the column, so as NOT introduce O2 to the column. Remove the insulator and the gray reducing nut that houses the gold seal and washer from the bottom of the inlet.

6. The injection port consists now of just a long metal tube. All flow lines and the glass liner have been removed from the assembly. Dip the gun brush into the Methylene chloride and insert the brush into the inlet completely. MOVE THE BRUSH UP AND DOWN DO NOT TWIST. Perform this step twice. Then use the glass pipette to rinse the inlet with Methylene Chloride. Follow the Methylene Chloride rinse with an Acetone brush and rinse and finish with a Methanol brush and rinse.

7. After the last Methanol brush and rinse, dab the top of the inlet with a “kimwipe” to
remove any residual solvent that may pool. Look down through the assembly to
make sure that it is free of particles that may stick to the walls of the inlet. Heat the
inlet to 65C to flash vaporize the solvent away. (THIS IS A CRITICAL STEP THAT
CANNOT BE BYPASSED).

8. Reassemble the inlet with a new gold seal, washer, liner, and a new ferrule on the
column. Make sure that the column is re-cut prior to installation. Reinstall the split
vent line and tighten. Reinstall the weldment assembly (NOTE: Prior to reinstalling the autosampler assembly turn the injection port flow back on. Do not turn the injection port temperature on.) Check to make sure that proper head pressure can be obtained, if not recheck all fittings for leaks. Before increasing the temperature, let the inlet sit at 65C for about 10 minutes to remove any oxygen that might be in the inlet. THIS STEP MUST NOT BE SKIPPED. IF IT IS THE CLEAN PARTS JUST INSTALLED WILL BE REOXIDIZED.

9. After 10 minutes heat the inlet to operating conditions. Let it stabilize for 5 minutes. Make at least 2 blanks runs before any analyte is injected, to make sure that the inlet has been successfully cleaned. Be aware this technique does not work on all applications, and over time will not bring the inlet back. The assembly will need to be changed in time.

Arsenic (As), mercury (Hg) element @ Varian 240FS

In general, for the Arsenic (As), mercury (Hg) element, you could use the flame AAS to detect the concentrations. However the detection limit is too high: for As, maybe 2-3 ppm; for Hg, maybe 2 ppm, {different instrument has different detection limits}. So the results make no sense = have zero practical meanings. That is the reason why V did not recommend using the Flame AAS.

For Arsenic: one of the main sources of interference in the determination of arsenic is the molecular absorption of flame gases and solution species at the extreme ultraviolet region of spectrum where the most sensitive lines for arsenic occur ( 93.7 nm and 197.2 nm). This non-atomic absorption can be corrected by using the deuterium lamp (D2) as a continuum light source. But do not expect too much on the D2 lamp correction.

For Hg: Hg ( I ) and Hg( II) show different sensitivities in the Air-Acetylene Flame AAS. In my opinion, suppose the Hg (I) is more sensitive due to the disproportionation reactions. Also for Hg at high concentration, there are always memory effects.

So for As, Hg: At low concentrations, these elements can be determined by vapor generation technique. That is why V recommend Hydride.

From my experience, I use following parameters.
As: NaBH4 0.6%, NaOH 0.5%, 5 M HCl, KI [ for As( III) is required ]
Hg: SnCl2 25% in 20% HCl, H2O.
For As, Hg testing: should use separate module to analysis for As, the other module for Hg or make 200% sure to clean the glassware to avoid contamination.

The Cleaning and Regeneration of Reversed-Phase HPLC Columns

A practical ways to return a contaminated column to — or close to — its original state.

What Causes Contaminant Buildup in Reversed-Phase Columns?
Usually, sample matrices contain compounds that are of no interest to analysts. Salts,lipids, fatty compounds, humic acids,hydrophobic proteins and other biological compounds are a few of the possible substances that can come in contact with an HPLC column during its use. These
materials can have lesser or greater retention than the analytes of interest.
Those compounds that have lesser retention, such as salts, will usually be eluted from the column at the void volume. These undesired interferences might be observed by a detector and appear as chromatographic peaks, blobs, baseline upsets or even negative peaks.

Washing Bonded-Silica Columns: The keys to rejuvenating a contaminated HPLC column are knowing the nature of the contaminants and finding an appropriate solvent that will remove them.
A recommended column washing system for a typical bonded-silica column and a mobile phase without buffer salts is to use:
• 100% methanol, • 100% acetonitrile, • 75% acetonitrile–25%isopropanol, • 100% isopropanol, • 100% methylene chloride and • 100% hexane.
When using methylene chloride or hexane, the column must be flushed with isopropanol before returning to an aqueous mobile phase because of solvent immiscibility. A minimum of 10 column volumes of each wash solvent should be passed through a column. For 250 mm x 4.6 mm analytical columns, analysts can use a typical 1–2 mL/min HPLC flow-rate. To return to the original mobile phase, chromatographers can usually skip going through the entire series in reverse order. Using isopropanol as an intermediate solvent is recommended, followed by mobile phase without buffer, then finally with the starting mobile-phase composition. Tetrahydrofuran is another popular solvent that can be used for cleaning contaminated columns. If users suspect severe fouling, they can mix dimethyl sulphoxide (DMSO) or dimethylformamide mixed 50:50 with water and pass them at flow-rates less than 0.5 mL/min. The successful regeneration of a reversed-phase column can be a time-consuming process, and solvent washings can be programmed into a gradient system for overnight operation.

Bonus Question: A question arises as to whether to reverse the HPLC column during the washing procedure? --- tips: This situation is particularly true if metallic ions are sorbed to the silica
or bonded phase. A chelating reagent such as 0.05 M ethylenediaminetetraacetic acid
(EDTA) can be flushed through a column. The EDTA complexes with many metallic
species and solubilizes them. After treatment with an EDTA solution, analysts
can wash the column thoroughly with water.

To control bacterial growth that could be present in a buffer system or in columns
left unattended in aqueous buffer, chromatographers can use common household bleach diluted 1:10 or 1:20 or 35% Nitric Acid . Run at least 50 column volumes followed by another 50 column volumes of HPLC-grade water. Do not run the bleach through the detector, because it could attack the flowcell.