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Sample injection system in hplc
1. Facilitators: -
Dr. B. M. Gurupadayya,
Dr. R. S. Chandan,
Dept. of pharmaceutical chemistry,
JSS college of pharmacy,
Mysuru.
Submitted by: -
Ram Mohan S.R.
1st M.Pharm
Pharmaceutical Quality Assurance
JSS college of pharmacy
Mysuru
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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2. INTRODUCTION
• The injection system is positioned after the pump head.
• The injection of a sample at atmospheric pressure into the system, at
high pressure, represents a critical step in the chromatographic
process.
• Sample injection valves, or switching valves, are used to introduce
reproducible amounts of sample into the HPLC eluent stream without
causing changes in pressure or flow.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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3. Contd……..
Injection System
• The injector is located on the high pressure side of the pump.
• Sample injection valves allow effective sample introduction without
interrupting the flow or altering the system pressure.
Components of the sample injection valve
• A needle or syringe to pierce the vial septum
• A metering device to measure the aspirated amount of sample liquid
• A loop or holding device to retain the sample prior to injection
• A valve which is used to alter the hydraulic path of the eluent
through the device in order to affect direct injection of the sample
plug into the eluent stream under pressure
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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6. Manual Injection Systems
• Manual sample injectors for HPLC transfer sample at atmospheric
pressure from a syringe to a Sample Loop. The loop is then connected
via a change in valve configuration, to the high-pressure mobile phase
stream, which carries the sample onto the column.
There are two methods of loading the sample:
• Complete-filling –where the loop size chosen has the desired injection
volume and is totally filled with sample
• Partial-filling –where the loop chosen is at least twice the required
sample volume and is only partially filled
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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7. Steps in a manual injection process.
There are mainly 3 steps
Step 1:
The sample is introduced using a
syringe into the sample loop via a port
on the injection valve. Depending upon
the injection type the loop will either
partially or completely filled.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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8. Step 2:
Once the sample is loaded the
valve is turned to the ‘inject’
position.
The rotor seal channels move and
connect different ports on the
stator. The pump is now able to
push mobile phase through the
sample loop, transferring the
sample onto the column.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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9. Step 3:
Once the injection has been made the valve is
returned to the ‘load’ position.
This enables loop filling for the next injection.
Some people prefer to leave in the inject position
until the next injection is required to completely
flush the loop and reduce ‘carryover’
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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11. Autosamplers
All autosamplers have the same basic components which include,
• The injection valve,
• A syringe or sampling needle,
• A loop of either fixed or adjustable volume,
• A metering pump to aspirate the sample from the vial and an
injection port through which the sample is introduced into the loop.
Most autosamplers use six-port loop injection valves in order to deliver
the sample plug to the analytical column.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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12. There are three main operating principles which are used in
autosampler design:
1. Pull-to-fill
2. Push-to-fill
3. Integral-loop autosamplers
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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13. Pull to Fill Auto Samplers
There are mainly 4 steps
Step 1:
This is the ‘inject’ configuration.
Mobile phase flows from the pump,
through the sample loop and into the
column.
The valve orientation effectively
isolates all other autosampler
components from the eluent hydraulic
path.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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14. Step 2 –Load:
The injection valve rotates to direct
the eluent from the pump straight
onto the analytical column.
The syringe or metering pump is now
used to PULL an exact volume of
sample from the vial and into the
sample loop this can be used in either
the full or partial loop mode.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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15. Step 3 –Injection:
The injection valve rotates again to
direct the mobile phase through the
loop –effectively displacing the
analyte band onto the analytical
column.
The rest of the autosampler is once
again isolated from the hydraulic
pathway.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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16. Step 4 –Wash:
The syringe and needle flow path through the valve are rinsed with a wash solvent to reduce the
amount of sample ’carry-over’ from one injection to the next.
This operation is carried out multiple times with a solvent in which the analyte and sample
components are highly soluble. The wash solvent is discarded to waste.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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18. Push to Fill Auto Samplers
There are mainly 4 steps
Step 1:
This is the ‘inject’ configuration.
As with the Pull to Fill configuration
the mobile phase flows from the
pump through the valve directly to
the analytical column, bypassing the
other autosampler components.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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19. Step 2 –Load:
The needle moves to the sample vial and the
metering pump is used to aspirate the sample into
the holding loop.
The injection valve switches and now the mobile
phase flows directly from the pump to the analytical
column.
This allows the metering device to PUSH the holding
loop contents into the sample loop.
The mobile phase which is displaced by the sample
is directed to waste via the valve waste port.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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20. Step 3 –Injection:
The valve switches and the
mobile phase is now directed
through the sample loop to push
the sample onto the analytical
column.
Once again the other
components of the autosampler
are isolated from the main
hydraulic path of the eluent.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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21. Step 4 –Wash:
A wash solvent may be used at this
point to flush any remaining
sample components out of the
system.
The analyte and other sample
components should be highly
soluble in the wash solvent.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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22. Integral Loop Auto Samplers
• In recent years, the integral loop autosampler has become popular.
The strong point of this design is that no sample is wasted and this
can be very important for trace analysis when sample volume is
limited.
• Because the sample is contained completely within the swept portion
of the loop, the entire volume of loaded sample is injected.
Additionally, continual flushing of the injection switching valve and
loop following injection helps eliminate sample carryover effects.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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23. There are mainly 4 steps
Step 1:
This is the inject configuration. The eluent
flows from the pump, through the valve
and into the syringe or metering device,
through the needle, the high pressure seal
and then towards the analytical column.
This configuration ensures that during
analysis, all major components of the
autosampler are flushed to reduce
potential carryover.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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24. Step 2 –Load:
The injection valve turns to the ‘load’ position, the
eluent flows directly from the pump to the analytical
column.
The autosampler components are now isolated from the
main hydraulic flow path.
The sample is moved into position, or the sampling
needle arm moves to the sample depending upon
design.
The sample is aspirated into the integral loop using the
metering pump or syringe device.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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25. Step 3 –Injection:
The valve switches to connect the
autosampler components into the
main hydraulic path.
The eluent displaces the sample
plug from the loop onto the
analytical column.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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26. Step 4 –Wash:
A wash solvent may be used at this point to flush any remaining sample
components out of the system.
The analyte and other sample components should be highly soluble in
the wash solvent.
Pharmaceutical Quality Assurance, Department of Pharmaceutics.
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