High Performance Liquid Chromatography System

GK Dwivedi, Madhu Dwivedi, Dhirendra Rathore, RS Chauhan & Gagan Sharma

Institute of Biotechnology
GB Pant University of Agri & Tech Patwadangar263 128 (Uttarakhand)

The HPLC (LC-20A) has been designed to provide high accuracy and high sensitivity
analysis. System configurations along with description of the operation of the various
components are provided below. Each component of the system is controlled locally for
stable analysis.
The low pressure gradient unit mixes upto 4 mobile phases that have been degassed by the
degasser
**

The mixer enhances the mixing efficiency of the mobile phases. This item is required
for low or high pressure gradient system.

**

The autosampler automatically injects the samples into the flow lines. By adding a
rack changes it is possible to automatically change autosampler racks.

Steps

Mobile phase is drawn out of the reservoir bottle and pumps through the tubing
by the pump.

The degasses removes dissolved air from the mobile phase preventing the air
bubbles and consequent rise drift or other baseline irregularities caused by
dissolved air.

Pump sends the mobile phase through injector, column and detector and finally
into the waste container.

Samples are injected into the system by injector with a syringe.

In the column, the components are separated by means of natural interactions of

the mobile phase and column packing (Stationary phase).

The detector detects the components eluted from the column and sends the
signal data to a chromato pacer computer.

Mobile phase from detector drains into the waste container.

Reservoir bottle

Degasser
Low Pressure Gradient Unit
Pump Unit
Mixer
Injector
Auto Samples
Column

Detector

Water Container

Column Oven:
It maintains the temperature of the Liquid Chromatography System column and flow
lines at a content temperature, in order to separate performance. The oven is an aircirculated thermostat chamber for HPLC. Column oven regulates temperatures with a heater
while AC model equipped with cooling mechanisms that enables it to regulate the oven and
flow lines to temperatures below room temperature. The air circulation ratio of the fan
automatically matches the set temperature to keep temperature constant. At high
temperatures, the air circulation ratio is increased to keep the temperature constant at
temperature close to the ambient temperature, the air circulation ration is decreased to

suppress heat generation due to air circulation. The fan speed is controlled so as to enable
stable temperature regulation.

Autosampler:
The applicable pressure in 20 MPa-35MPa
The injection capacity ranges from 0.1l to 2000 l
There re two modes for samples injection
Standard injection mode that facilitates condition setting. This
is the simplest mode. In this mode, specify the sample vial
number, number of injections, injection volume and analysis
time in sample table setting screen. Set needle stroke, rinse
volume, rinsing speed, sampling speed in parameter setting
Bracket sequence analysis mode that insertion analysis can
be performed in a certain cycle. This mode can be used to
inject a predetermined number of samples repeatedly at
periodic intervals.

Sample Injection: A number of steps are followed:

Stand Mobile phase from the reservoir is pumped through high
pressure valve, sample loop, sampling needle injection forth
and back through high pressure valve before reaching the
analysis column.
Release of pressure in flow line High pressure
valve rotates to 600 clockwise position. Under the
high pressure in sample loop mobile phase remaining
in the sample loop flows through the sample needle,
sample loop, high pressure valve, measuring pump,
low pressure valve and rinsing port, relieving the
pressure in sample loop.
Rinsing of sample needle before sample aspiration,
Low pressure valve rotates to 300 clockwise position.
Sample needle is inserted into rinsing port where
rinsing is done with the rinse solution.

 Sample aspiration The sampling needle is inserted into

sample vial, measuring pump draws the sample into the
sampling needle and sample loop.
Rinsing of sampling needle, sample aspiration.
Start of analysis The sampling needle is inserted into the injection port. High
pressure valve rotates 600 counter clockwise to the injection position.

Selection of Rinse Solution

Depending on mobile phase select the rinse solution
Methanol: Water (50:50)
When target compound is acid, base or conic substance, add formic acid and acetic
acid to organic solvent (methanol, acetonitrite) or 0.1% TFA solution or mixture.
For non aqueous phase, use same solvent as mobile phase.
Use 0.1% TFA aqueous solution, organic solvent or mixture of both when acid, base
or tonic substances are the target compound.

Preparing Samples
Completely dissolve the sample in a solvent with an identical or similar composition
to mobile phase.
Filter the sample to eliminate undissolved particles to avoid clogging the needle.
Dilute highly viscous sample before use as they as not aspirated.
Carefully load the sample into a vial by injecting.

Setting Samples in the Sample Rack

Upper surface of the rack is marked with numbers at the vial positions. Specify these
numbers when setting sample number.

Setting Sample Numbers

Open the door
Place the vial in the sample rack with cap facing up.

 Slide the sample rack. The sample rack check into place when it is inserted correctly.
(If the sample rack is not inserted correctly, the sampling needles may stick the
wrong position and may be bent.
Close the door.
The sample is injected along with the mobile phase into the flow
lines passes through the high pressure valve and finally into column
where analysis begins. Purge of measurement flow line (Rinse
solution aspiration). The low pressure value rotates 300 counter
clockwise. Plunger of the measuring pump has returned to original
position, then the low pressure value rotates 600 clockswise the
plunger operates to aspirate rinse solution.
Rinse Solution Dischargement
The low pressure valve rotates 600 counter clockwise measuring
pump plunger operates to discharge rinse solution to rinse port.

Sample Vials Racks

1ml sample vial rack

175 vials

1.5 ml sample vial rack

105 vials

1.5 ml sample vial cooling rack

70 vials

4 ml sample vial rack

50 vials

Sample Vials
4ml sample vial

4ml cap

borosil glass

1.5 ml sample vial

1.5 ml

B Glass

1.1 ml sample vial

1.1 ml

B Glass

1 ml sample vial

1 ml

Polyethylene

1ml sample vial

1ml

Cap

Polyethylene

--

--

Both glass and plastic (polypropylene) sample vials are used. In case
of glass vials, the ionic substances, acids or bases may be adsorbed onto glass surface.

Thus yield is poor and reliability of the analysis is lost. To avoid the problem of adsorbtion
use:
10-100 mM aqueous Perchloric acid solution or mixture of Perchloric acid + Organic
solvent (acetonitrite, methanol, ethanol).
Organic solvent solution of 10mM trifluoroacetic acid (TFA).
Use Polyethlylene vials.
Some times in plastic sample vials, the hydrophobic properties of substances cause
surface adsorbtion results into poor results and reliability. Use mixture of water or buffering
solution with organic solvent (20:50 v/v).

Degasser
Degasser is a device removes
dissolved gases from liquids using
a

special

membrane.
degasser

synthetic
By
to

polymer

connecting
solvent

the

delivery

system, gasses dissolved in mobile phase.

Degasser prevent the formation of gas bubbles caused by dissolved gases.
It improves the stability and reproducibility of HPLC analysis.
The mobile phase needs to be degassied passes through a special synthetic polymer
membrane inside the vaccum chamber. The dissolved gas has a smaller molecular
size and higher mobility has strong affinity for the polymer membrane. It permeates
though the membrane into vaccum chamber is discharges from the degasser and
thereby eliminated from the solvent.

There are Two Methods for Degassing Mobile Phase:

1.

Degassing by pressure reduction and synthetic polymer membrane.

2.

Bubbling helium gas in a mobile phase in the reservoir and driving out the
dissolved air.

Following flow rates are used to avoid bubble formation.

Water/methanol

1.5 ml/min

Water/acetonitrite

1.7 ml/min

1.

Spectrofluorometric Detector RF-10AXL

2.

Differential Refractometric Detector : RID-10A

3.

UV/VIS Photodiode Array Detector: SPD-M2OA

Spectrofluorometric Detector RF-10AXL

Highest Sensitivity of the detector
Highest wavelength accuracy
Highest wavelength reproducibility

Differential Refractometric Detector RID-10A

Principle of Measurement
The light radiated from the lamp passes through the lens slit and cell. The rays are reflected
by the mirror and passed through the cell to form an image of the slit on the photodiode. The
cell consists of the sample side and reference side. When the refractive index (RF) in
sample side cell varies, the image of slit moves horizontally. The photodiode unit is
partitioned unit is partitioned into four units. The unit provides measurements for both
analytical and preparation work.

 The signal is processed using the right and left portions of the photodiode as
individual elements. Data processing is performed as a two partitioned
photodiode divided into right and left. When refractive index in the cell varies, the
balance of the incident light intensity into right and left parts of the photodiode
changes. This change is converted into refractive index and can be recorded.
Signal processing is performed using upper and lower parts of the photodiode
elements. The refractive index is one-twentieth of mode A.

UV/VIS Photodiode Array Detector: SPD M20A

The photodiode array continuously monitors spectra over the entire wavelength
range 190-800nm. Both deuterium and tungsten lamps (D 2 and W) as light sources allows
high sensitivity measurement of chromatograms and absorbance spectra over wide
wavelength range of 190-800 nm.
Continuous monitoring of the absorbance spectra of eluted components provides
valuable information for an analysis:

Ide
ntifi
cati

on of component from absorbance spectra.

Use of retention time Rt along with absorbance spectra provides greater certainty in
anlayte identification.

Check of impurities (Eluted peaks can be checked to determine if they are generated
form single component or mixed with impurities.

Wavelength accuracy is checked automatically from UV to vis wavelength range

using the holmium oxide filter absorbance spectrum and deuterium (D 2) emission line
spectrum.

Upto 4 wavelengths can be output using multi-wavelength UV detector.

D2 lamp illumination only 190-600 nm
W temperature illumination only 371-800 nm
D2 + W simultaneous illumination 190-800 nm

Temperature of Flowcell:
If ambient temperature is 20-300C, set cell temperature to 400C
If ambient temperature is 30-350C, set cell temperature to 500

Fraction Collector FRC-10 A

The fraction collector is a device to collect the fraction of a sample for further analysis
using either MS.NMR. X-rays and crystallation graphic analysis.

The device is controlled by system controller.

For 4 ml volume 144 fractions and 64 fractions are used for large fractionation.

Fractions are cooled at 40C in the system.

The delay time from the detector to fractionation head is automatically calculated. It is
not necessary to calculate
it every time the flow rate is
changed.

10

When exceeding the volume of the vial, the collector automatically moves to the next
vial before it is overflown. So there is no worry for the loss of precious fraction.

Collection Method
There are two methods:

Fractionation head with valve using valve.

Fractionation head using tubing.

Unnecessary liquid in analysis if flowed in the drain tubing so that test tubes are not
wasted.

Liquid is not dispersed in moving from one test tube to another.

Liquid from the previous fractionation remains between inside of the valve and nozzle
tip 50 l. Therefore, a small amount of contamination may occur. Time programme is
set.

Contamination does not occur since liquid is collected into test tubes from the tube
connected to the detector or cell without passing through the flow line selector.

Liquid may be dispersed in moving from one test tube to another when the flow rate
is high.

11

System Controller CBM 20A

This instrument consists of networking capability to set HPLC system parameters to
create analysis conditions, execute analysis and monitor the system from PC. It is
also possible to process the data from remote PC.
It controls pump (delivery module), autosampler, column over, Detector and fraction
collector.
It can control the photodiode array detector as a 4 wavelength detector.

12