Nanospray Tips & Tricks

Troublshooting Clogs

When it comes to basics, a nanospray emitter is simply a hollow glass tube. Granted, the inner diameter is no bigger than the size of a strand of hair, but it is essentially a glass tube. Clogs are formed when foreign matter is introduced into this very tiny space and becomes trapped in the inner taper of the tube where the tip is formed. These foreign particulates can be generated by many things, and they need not be very large to have a big impact on performance.

Clean, clear tubing

All New Objective pre-packed columns, self-pack columns, and emitters are both visually and machine inspected prior to shipping, and are guaranteed to have an open bore that is clean and free of obstruction. Because we go to great lengths to provide this guarantee, clogging is caused by improper use, and does not fall under our manufacturer's warranty.

Most clogged column and/or tip problems can be easily diagnosed with visual examination using a transmitted light microscope (10X). You just need to understand what to look for.

Following are the three most common reasons for clogging. Following a few simple best practices when using nanospray components will greatly reduce the chance of clogging, and increase success.

Contamination in water

Problem: Black bands of material at the head of the column

Although your in-house system may be state-of-the-art, it's only as good as the maintenance on the filters. Using in-house meg-ohm water can cause carbon particulates to build up within columns. Even when filters are maintained, carbon particles can be released into the system. These carbon particles will find their way into your column. (Remember that this tubing ID can be smaller than the diameter of your hair.) Carbon build-up will often result in a black band at the head of the column.

Best practice: HPLC Distilled-in-glass bottled water
Using HPLC-grade bottled water reduces the likelihood of clogging due to particulate contamination.

Fused-silica particles

Problem: Chunks of "glass" in tip or at column head

Improper cleaving of fused-silica tubing can lead to silica shards in your column or tip. A poorly cleaved piece of tubing, when inserted into a fitting and twisted into place creates shards and particles of fused-silica. These particles not only become embedded into the fittings, but can also be introduced into the flow path. These particles will usually appear transparent and have sharp, fractured edges.

Best practice: Use proper cleaving tools for capillary tubing
Always be sure to follow proper cleaving procedures and cleave the end of all columns and fused-silica tips prior to use. Use diamond- or sapphire-tipped tools to ensure clean scoring and cleaving. Ceramic wafers are not considered sharp enough for capillary tubing, as they dull quickly and cause ragged ends. Contaminated filters and unions should be replaced immediately. Cleaning cannot guarantee that they are contaminant-free.

Poorly packed column

Problem: Unidentified material in column bed/in emitter

Column packing requires patience and practice. It is very easy to allow contaminants and foreign particles—from unclean tubing, poorly stored media, degraded fittings, residual media external to frit—to be lodged in your column bed during the packing process.

Additionally, emitter clogs can be formed from chromatogrphy media "bleeding" from the column and collecting in the tip. The media collects and is wedged into the taper, creating a very effective clog.

Best practice: Use bead-fritted tubing for packing/use pre-packed columns
Using properly fritted tubing will help reduce column bleed issues when self-packing, as a beaded frit will not allow media through, regardless of the particle size. New Objective offers quality pre-packed columns, which are all inspected prior to shipment. To ensure bleed-free columns that are also consistent and contaminant-free, use professionally-packed columns. New Objective can also custom pack columns with customer-supplied chromatography material. Contact us to find out more.

Configuring a Sample Valve

The IntegraFrit Sample Trap column can be easily integrated into your sample injection system. It consists of an IntegraFrit column, packed with 2.5 cm of any of our in-house media—or customer-supplied media— and the Upchurch Sample Trap cartridge.

IntegraFrit Sample Trap plumbed into valveSimple valve configuration for sample trap application

In a very common configuration, a sample injection is integrated into sample loop (A) and flushed into the IntegraFrit Sample Trap (B).

The sample is consequently concentrated and purified. With a valve switch, the trap is then placed inline with a PicoFrit analytical column (A) for gradient elution.

Loading Sample into Offline GlassTips

The optimal taper shape of GlassTips makes sample filling a snap, featuring a "self-filling" design. Normally troublesome air bubbles (which can cause "vapor lock" in other tips) have no negative effect on performance.

Sample loaded in pipette

Pipette inserted into GlassTip

Gently push sample into GlassTip

Sample fills tip via capillary action

Load your sample into a suitable pipette tip. Shown here is a 2 µL sample in a recommended GELoader pipette tip, using a 0.1 to 10 µL pipettor.

Insert the GELoaderâ„¢ tip into the distal end of the GlassTip. Press slowly on the plunger to inject your sample. This is important with viscous peptide and protein samples. To accommodate the sample introduction, slowly withdraw the GELoader from the emitter as the pipetter is squeezed.

As seen in the photo, the sample has been loaded into the far (distal) end of the tip. Capillary action will guide your sample into the tapered region of the emitter. There is no need to centrifuge your sample down into the nanospray tip.

Within a minute or two, capillary action has "pumped" your sample into the tip region. The GlassTip is now ready to mount on your nanospray source.

Storing PicoFrit Columns

f you need to store a PicoFrit® Column for reuse on a later date, we recommend the use of a MicroTight™ union, fitting, and sleeves to protect the tapered end of the column. Letting the column dry out for an extended period of time may render it unuseable. Protecting the tip end will allow you to store the column in a solvent-filled vial, beaker, or centrifuge tube.

Microtight union and sleeve

PicoFrit column inserted in union

Gently withdraw tip inside sleeve

Store union in solvent-filled vial

Slip the distal end of the PicoFrit column into the MicroTight sleeve, and pull through. DO NOT feed the column tip end first!

Pull the column further into the sleeve so that the tip is fully covered and protected by the sleeve. Tighten the fitting so that the column tubing is firmly held in place.

Place the column into a suitable vial or centrifuge tube and fill the container with suitable mobile phase. (The same composition used for column conditioning is recommended.) For best results, the column should be vertical, with the tip pointed down. For long-term storage, the container should be covered with a suitable material to retard solvent evaporation.