High performance liquid chromatography basics

1. COMPONENTS AND
APPLICATIONS

2. High-performance liquid
chromatography (HPLC; formerly referred to
as high-pressure liquid chromatography), is a
technique in analytical chemistry used to
separate, identify, and quantify each
component in a mixture.

3. 1.solvent storage bottle or reservoir
2.pump
3.Injector
4.Hplc column
5.Detector
6.Chromatogram

5.  The solvent mixture of the solvents used as the
mobile phase. The solvent stored in it pump
into the column to perform sepration in it.

6. The role of the pump is to propel (force) a
liquid (the mobile phase) through
the chromatograph at a specific flow rate,
expressed in ml/min. Normal flow rates in
HPLC are 1-2 ml/min and typical pumps can
reach pressures in the range of 2000 – 9000 psi
but in applications covered under UHPLC
mode operating pressure can be as high as
15000-18000 psi. The normal flow rate stability
must be < 1%.

7. CONSTANT PRESSURE
PUMPS
CONSTANT FLOW
PUMPS
Provide consistent
continuous flow rate
through the column
with the use of pressure
from a gas cylinder.
a) Reciprocating Piston pumps
deliver solvents through
reciprocating motion of a piston in
a hydraulic chamber. The main
drawback of a reciprocating pump
is that it produces a pulsing flow.
With a flow-sensitive detector,
such as micro-adsorption detector,
a pulse damping system must be
used and detector sensitivity is
reduced. b) Syringe type pumps
are suitable for small bore
columns. Constant flow rate is
delivered to column by a
motorized screw arrangement.

8. ISOCRATIC PUMP GRADIENT PUMP
It delivers constant mobile
phase composition
delivers variable mobile
phase composition

9. the sample should be introduced as a plug,
without disturbing the column packing. The
injector serves to introduce the liquid sample into
the flow stream of the mobile phase. Typical
sample volumes are 5-20 μL. The injector must be
able to withstand the high pressure of the mobile
phase. A pre column, a small removable section of
tubing containing the same packing material as the
column, can be used ahead of the analytical
column to protect the latter from contamination.
The pre column also acts as a buffer to prevent
channeling of the packing during injection.

10. The column is considered the heart of the
chromatograph. The success or failure of a
particular analysis depends on the choice
of column. The column’s stationary phase
separates the sample components using various
physical and chemical parameters.
There are four types of columns used in HPLC:
High performance analytical columns [internal
diameter (i.d.) 1.0-4.6 mm; lengths 15 –250 mm] -
used mainly for qualitative and quantitative
analysis Preparative columns (I.d. > 4.6 mm;
lengths 50 –250 mm) – used mainly for preparative
work Capillary columns (i.d. 0.1 -1.0 mm; various
lengths) Nano columns (i.d. < 0.1 mm)

11.  ION EXCHANGE HPLC COLUMN
 Ion exchange and ligand exchange
chromatography may be combined in a
column. In these combined-mode columns, ion
exchange is usually via metal ions, and the
ligands are electron-donor molecules such as
hydroxyl groups or amines. This type of HPLC
column is frequently used for the separation of
monosaccharides

12.  LIGAND EXCHANGE HPLC COLUMN
 Ion exchange and ligand exchange
chromatography may be combined in a
column. In these combined-mode columns, ion
exchange is usually via metal ions, and the
ligands are electron-donor molecules such as
hydroxyl groups or amines. This type of HPLC
column is frequently used for the separation of
monosaccharides.

14. The detector is design to hold a relatively small
volume (usually 5 to 10 micro liter) and is
placed near the exit side of column as possible
the electrical signal from the detector is
amplified, if necessary and routed to a
recorder which record to developed
chromatogram. Effluent from the detector is
collected in a suitable container for later use or
is discarded

15. The chromatogram is a graph that monitors the signal in the detector over time.
As chemicals are detected by the instrument, the signal increases, and
the chromatogram displays a "peak." ... Each peak is labeled with retention time.
Retention time indicates how long it takes for a compound to come out of the
HPLC column.

17. As briefly mentioned, HPLC has many applications in both
laboratory and clinical science. It is a common technique
used in pharmaceutical development, as it is a dependable
way to obtain and ensure product purity. While HPLC can
produce extremely high quality (pure) products, it is not
always the primary method used in the production of bulk
drug materials. According to the European pharmacopoeia,
HPLC is used in only 15.5% of syntheses. However, it plays
a role in 44% of syntheses in the United States
pharmacopoeia. This could possibly be due to differences in
monetary and time constraints, as HPLC on a large scale can
be an expensive technique. An increase in specificity,
precision, and accuracy that occurs with HPLC
unfortunately corresponds to an increase in cost.

18. Similar assays can be performed for research
purposes, detecting concentrations of potential
clinical candidates like anti-fungal and asthma
drugs. This technique is obviously useful in
observing multiple species in collected
samples, as well, but requires the use of
standard solutions when information about
species identity is sought out. It is used as a
method to confirm results of synthesis
reactions, as purity is essential in this type of
research. However, mass spectrometry is still
the more reliable way to identify species.

19. Medical use of HPLC can include drug analysis, but falls
more closely under the category of nutrient analysis. While
urine is the most common medium for analyzing drug
concentrations, blood serum is the sample collected for most
medical analyses with HPLC. Other methods of detection of
molecules that are useful for clinical studies have been
tested against HPLC, namely immunoassays. In one
example of this, competitive protein binding assays (CPBA)
and HPLC were compared for sensitivity in detection of
vitamin D. Useful for diagnosing vitamin D deficiencies in
children, it was found that sensitivity and specificity of this
CPBA reached only 40% and 60%, respectively, of the
capacity of HPLC. While an expensive tool, the accuracy of
HPLC is nearly unparalleled.
See also