HIGH PRESSURE LIQUID

CHROMATOGRAPHY
High pressure liquid chromatography (HPLC) refers to liquid chromatography that is done under
high pressure. The sample is introduced into the system via an injector. The injector may be
manual or automatic. This type of sample introduction via the injector is however much more
complicated than the ones applied in gas chromatography as injection via the use of a syringe
alone is not possible. It involves a 6-port rotary injection valve that is specially designed to serve
this purpose. The function of the syringe is to put the sample into the high-pressure flow in as
little volume as possible in order for the sample to enter the column as a homogeneous, lowvolume plug.

MANUAL INJECTION
The introduction of the sample is done at atmospheric pressure and is passed into a constant
volume loop after it has left the syringe. At this point the loop is in the load position and has not
yet come into contact with the mobile phase as yet.1 However, it comes in contact with it through
rotation (as the name suggests).1 It is the rotation of the injector position that brings the loop in
contact with the mobile phase after which the sample is lead into the column. In order to ensure
that there are no air bubbles present or that there are no previously used sample left behind, some
of the sample is allowed to run into a waste loop.1

AUTOMATIC INJECTION
This form of injection is a more preferred one as it eliminates personal error and also improves
the productivity of the lab. This type of injector, as the name suggests, has both an automatic
injector and an automatic sampler.1 The filling of the loop and delivery of the sample is
controlled by software programs.1The sequence of samples for injection from vials kept in

numbered positions of the auto sampler is also controlled by the computer. Some of the
precautions that apply to this system are:
i)

In order to prevent the transfer of left over samples from one injection to the next the

ii)

needle is washed between samples.

It is ensured that the tubing is rid of all previously used solvents or buffers (by
washing) before the start and at the end of the analysis.1

There are four types of injectors used in HPLC; namely, type 1, 2, 3 and 4 as shown in the figure
below. 4

TYPE 1
When using the type one injectors, completely filled sample loops are used in order to determine
the volume injected. A volume of sample is either pushed or sucked into a sample loop by a
syringe fill it completely. The injections obtained are very precise because it is the loop volume
that determines the volume injected.4

TYPE 2
This type of injector uses a micro syringe to transfer sample into the loop. Also, in this type of
injector the sample size is always smaller than the loop volume. So it is the syringe that
determines the loop volume in this case. The precision is not as in in this type as it is in type one.
However, no sample is trapped or wasted in injector type.4

TYPE 3
This type of injector uses both the complete and the partial filling methods. In this type, the loop
is loaded by inserting the syringe into the needle and then dispensing it the contents. The syringe
is allowed to remain in the port until the valve has been switched. This switching action allows
the loop to be inserted into the stream without the exposure of the syringe to high pressure. The
syringe is removed while in the injection position. As a result some of the sample is trapped in a
connecting passage of the injector.4

TYPE 4
This type of injector also uses both methods, except that no sample is trapped in this one. The
other major difference between this type and the type 3 injector is that it does not have a
connecting passage between needle tip and sample loop.4

ADVANTAGES OF INJECTORS IN HPLC

Some of the advantages of the manual injector used in HPLC are:

1) When the syringes used in HPLC are compared to those used in gas chromatography they
can be considered relatively easy to use.2
2) The volume of samples used can be varied quite suitably by changing the loop.2
3) The sample deliveries made are very reproducible.2
4) They are made to withstand high pressure due to their construction using stainless steel.2

DISADVANTAGES OF INJECTORS IN HPLC

Some of the disadvantages of using the manual injector in HPLC are:
i)

This can be consider quite a complicated process and would therefore require a

ii)

trained technician. Thus this can be seen as a disadvantage.

Also because of the fast process it has low sensitivity towards some compounds.

Table 1: Applications of Injectors used in HPLC3

Description
6-port centered injector

Fittings

1/16 Valco

Applications
Microbe HPLC
Analytical HPLC

References:
1) http://lab-training.com/landing/free-hplc-training-programme-12/
2) Dr. Vaughn Rattery lecture notes.

3) http://www.vici.com/cval/cval_oem_inj.php
4) www.itim-cj.ro/PIM/2003/2001/Volum/PosterIzotopi/PI20.doc