But with this separation power comes some limitations: 1) Capillary
columns, because they have smaller diameters (0.05 to 0.53 mm) than packed
columns (i.d. 2 to 4 mm), require relatively specialized injectors and ancillary
flow and pressure controllers and 2) capillary columns require a smaller
amount of sample than packed columns. While the average sample mass of
each component in a mixture that is separable by packed column GC can be
in the microgram range (10-6 grams) per injection, capillary
columns routinely only handle 50 nanograms (10-9 grams) of a
particular component or less, sometimes much less.
An Overloaded Chromatogram
The following figure shows a little better chromatography with fewer overloaded peaks. The second eluting peak (about 6 minutes) is clearly not overloaded while the group between 10 and 14 minutes still shows overloading characteristics: long drawn-out tailing and much less than baseline separation with peaks that elute nearby (the 11 and 12 minute peaks, for instance).
Schematic of packed GC column injector with septum purge
One last subtle point about the configuration of the carrier gas inlet: notice that it enters the injector at about the middle of the heating block and its gas has to travel along the outside of the injector before it enters the injection port liner AT THE TOP. This is so the carrier is preheated before it enters the liner where the sample is vaporized. This helps to prevent a cold spot at the top of the injector where the carrier gas enters.
Now remember that the size of the capillary column limits the amount
of analyte that can be injected, otherwise, chromatographic overloading
occurs. Therefore this packed column injector design, if used with a capillary
column, would require that samples with high concentrations of analytes
be diluted. Unless... what other alternative is there to get the amount
of analytes that are injected onto the column smaller without having to
dilute concentrated samples? The solution is the split/splitless capillary
A very neat (and fundamental) aspect of this is that the amount of gas exiting the split vent can be varied while keeping the flow onto the column constant. This means that the AMOUNT of the split (called the split ratio) can be varied (in modern instruments via software control). A common split ratio is 50 to 1. That is, for every 50 units of gaseous sample that are thrown away to waste, 1 unit goes on (into) the column.
The analyst keeps careful control of the split ratio so that results from the chromatography can still be quantified. Using a split ratio of 50, chromatographic peaks that show up as, say 2.5 ng of compound X (using a calibration curve of detector response versus peak area) really represent 2.5 x 50 = 125 ng of analyte X in the original sample (the split ratio was 50 remember?). Also notice that this analyte mass (125 nanograms) would have overloaded the column if all of it ended up on the capillary column. Voila! A split injection, and no sample dilution required.
A mode of injection that avoids the hot injection liner all together is useful for thermally unstable (labile) or for GC samples with large, analyte-boiling-point differences that suffer discrimination in flash vaporization. This is the cold on-column injector. Here's one design: In this specialized set-up (easily available via retrofitting common split/splitless injectors) a tall, low-thermal-mass extension is attached to the top of the injector. This keeps the needle guide cool from heat arising from the GC oven at the top of its temperature program. The needle guide ends in a valve system with a duck-bill septum at the top of the normal split/splitless injector whose glass insert has been replaced with a special insert that funnels the conclude needle directly down into the GC column.
The normal, metal syringe needle is replaced with a small diameter section of capillary tubing. The capillary "needle's" outside diameter (e.g., 0.15 mm o.d.) is smaller than the inside diameter of the GC capillary column (e.g., 0.32 mm i.d.) and the tubing is very long (~18 cm) in order to pass through the needle guide extension, the duck-bill valve/septum, the replacement insert, and into the first few centimeters of the column.
The on-column injection goes like this: A liquid sample is drawn into the on-column syringe. The opening of the duck-bill septum—and stopping of carrier gas flow—is initiated by pressing down on the needle guide which splits open the duck-bill septum's "lips". The syringe's capillary needle is inserted down through the needle guide, through the opened duck-bill septum, completely through the injector—whose heater is off since it's no longer needed to vaporize the sample—and into the GC column itself. The liquid sample is injected directly into a cool column ("on" the column); the capillary needle is withdrawn; the duck-bill septum closes when the needle guide is released; and carrier gas flow is reestablished. Now the oven's temperature program is begun and the sample vaporized to begin chromatography, never having come in contact with the reactive walls of a hot injection liner.