LC Instrumentation-2024

Liquid Chromatography-
Instrumentation
Department of Analytical Chemistry, Faculty of

Chemistry, University of Science - HCMC
Assc. Prof. Nguyễn Ánh Mai
2024
• In LC separation based on differences in
affinity with SP and MP
• LC formats: column/thin layer/High

performance chromatography
LC formats
• Column chromatography Class separation

Purification
Preparation
• Thin Layer Chromatography (TLC)
Analytical
• High Performance
Liquid Chromatography Preparative
LC FORMATS – column chromatography
Column chromatography
for
- Preparation
- Fractionation/
class separation
- Purification
Thin layer chromatography
Separation of black ink on a TLC plate

Thin-Layer
Chromatography
RF = b / a
Thin Layer Chromatography (TLC)
Flow through porous layer  CAPILLARY FLOW
Capillary force drives mobile phase up through TLC plate

Detection in TLC: plate with fluorescence
indicator or spraying reagents
Ninhydrin as reagent for primary amine

High Performance TLC (HPTLC)
•fine particles (5-6m)
•automated sample application
•instrumental detection
Comparison of the separations of amino acids on
classical TLC and HPTLC under identical
conditions.
• sharper zones
• shorter migration distances/faster
analysis
• twice the number of samples
simultaneously
Classical TLC plate (10-12 m) HPTLC plate (5-6 m)

High PerformanceTLC :
Densitometer
Separation of steroids *
Silica gel 60 HPTLC plate; 15 min development;
MP: C6H14:CHCl3: CCl4:C2H5OH = 7:18:22:1
Thin Layer Chromatography (TLC)
Advantages Disadvantages
• No instrumentation, • Low resolution
portable • Problems with
• Inexpensive controlling mobile phase
• High-throughput screening velocity and composition
• Crude samples (disposable • Require other
stationary phase) techniques for
• Easy to learn confirmation of solute
• Qualitative, semi- identity
quantitative • Qualitative, semi-
• Lab-scale preparative quantitative
• ….
HPLC system
System control &
data processing
Detector
Injector
Column
Chromatogram
Pump
Eluent
Particulates and dissolved gases in MP
cause problems
• Particulates can block the solvent reservoir inlet
filter, score piston, wear piston seals and block
column inlet frits (leading to high back pressure)
 MUST be removed by filtering through 0.45 or
0.22 m filter membranes
• Gas bubbles from dissolved gases can change tR,

distort peak shape or increase baseline noise
 solvents MUST be degassed before use
Filter membrane
Filter membranes should be compatible with solvents

Solvent-filter membrane compatibility
Filtering solvents
Syringe filters for sample solutions
Filters for solvent reservoir
Column frits
Plugged frit Clean frit

Injector Online filter Guard column
Mechanical +
chemical filter
Analytical column
Problems from gas bubbles
In pump In separation column In detector

Dissolved gas removal
- Sonication (less effective)
- He/N2 sparging (most effective)
- Vacuum
(online-degasser)
Effects of Temperature on Quantity of Dissolved CO2, O2, N2, and He
(at partial pressures of 1 atm)
Online
degasser
MP delivery system-reciprocating pump
Flow rate determined by piston diameter, length

of travel, and frequency of pumping
10-400 L/stroke
5-100 L/stroke
check valve a)
plunger head
b)
sub plunger
main plunger
Pump with 2 parallel connected plungers
• Low pressure pulse

• High accuracy
• Suitable for low volumes
(L- nL)
Pump with 2 plungers in series
Piston volumes are different!

High pressure pump
(mixer behind pumps)
Low pressure pump

(mixer in front of pumps)
Low pressure pump High pressure pump
Susceptible of buble forming Less susceptible of buble

because solvs. are mixed at forming because solvs. are
atmospheric pressure mixed at high pressure
• Compessibility & volume

No error due to volume change change effect accuracy
Less solv. compressibility effect
• Separate pump for each
solv.
Pump
Column Restrictor
Mixer
If restrictor is connected downstream from the detector, a little

pressure applied from the detector to prevent air bubles in detector
Kenics static mixer
Pressure pulse from pump
GOOD
The noise from pump contributes to the total noise of the system
BAD
PULSE - DAMPER
out
diaphargm in
The most usual damper type is a membrane one, The left

part is filled with inert liquid (heptane). Compressibility of
this liquid is enough to compensate for the pulsations of
the dual piston pump with piston volume around 100 L
• Flow reproducibility:
to collecting MP for certain periods of time
• Composition accuracy:
step height increase for mixing 2 solvents
the largest variations in composition accuracy can be

expected for solvent mixtures containing only a few % of
one of the solvents
Mixing solvents
Miscible
Immiscible
Temperature dependent
-> careful with solv. composition
close to the solubility limit
6-port valve injector for sample introduction
Sample loop
(2, 5, 10, 20 µL)
LOAD INJECT
Sample volume to be injected
Fully filled
Detector signal
Partially filled
1/2 x loop vol. 3 x loop vol.
Injection volume
> 3-5 loop volume < 1/2 loop volume

for fully filled for partially filled
DECTECTORS OF HPLC
• UV / DIOD ARRAY
• FLUORESCENCE
• REFRACTIVE INDEX
• MS DETECTOR
UV detector flow cell
from column
• Avoid using alkaline
solutions with pH > 9.5
• If unused overnight →
at least > 10% organic
mobile phase to prevent
algae growth.
• Flow cell volume should not

be larger than about 10% of
the peak volume
to waste
Flow cells using optofluidic waveguides (Agilent)
Total internal reflection gives

almost 100% light transmission
Long path length High S / N

Small cell volume
Diod Array Detector
(DAD)
• Allows for the best

wavelength(s) to be
selected for actual
analytes
• Peak purity
by absorbance
rationing at several
wavelengths
Chromatogram with DAD/PDA detector
Absorbance
Time (min)
Wavelength (nm)
Spectra as extra information for analyte identification

Peak purity checking with DAD/PDA detector
UV cutoff
• UV cutoff: the wavelength at which the absorbance
of the solvent in a 1cm cell (versus air as reference)
is equal to 1
• A solvent with UV cutoff higher than the working

wavelength causes high a high background absorbance
Beer’s law A = log (Io/I) = ×l × C
A: absorbance
Io : incident beam intensity
I: transmitted beam intensity
: molar absorptivity
l: cuvette length
C: concentration
UV spectra of
common reversed-phase solvents
Methanol
THF (UV)
Acetonitrile
Carbamate pesticides separation on C18 column. UV
detection,
Gradient: MeOH 35 – 65%
UV cutoff
Common additives in mobile phase for HPLC-MS
• Trifluoroacetic acid
• Formic acid
• Acetic acid
• Ammonium hydroxide
• Triethylamine
• Ammonium bicarbonate/formate/acetate
Impurities in solvents and additives in mobile phases
should also be considered in the UV cutoffs!
Trifluoroacetic acid
Acetic acid
Phosphoric acid
Wavelength (nm)
Viscosity
• Column backpressure P = 1000 (LF / r2dp2)
• Increase in viscosity → decrease column efficiency

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Liquid Chromatography-

Department of Analytical Chemistry, Faculty of

• LC formats: column/thin layer/High

• Column chromatography Class separation

Separation of black ink on a TLC plate

Flow through porous layer  CAPILLARY FLOW

Capillary force drives mobile phase up through TLC plate

Ninhydrin as reagent for primary amine

Classical TLC plate (10-12 m) HPTLC plate (5-6 m)

• Gas bubbles from dissolved gases can change tR,

Filter membranes should be compatible with solvents

Plugged frit Clean frit

In pump In separation column In detector

- Sonication (less effective)

- He/N2 sparging (most effective)

Flow rate determined by piston diameter, length

• Low pressure pulse

Piston volumes are different!

Low pressure pump

Susceptible of buble forming Less susceptible of buble

• Compessibility & volume

If restrictor is connected downstream from the detector, a little

The most usual damper type is a membrane one, The left

the largest variations in composition accuracy can be

1/2 x loop vol. 3 x loop vol.

> 3-5 loop volume < 1/2 loop volume

• Flow cell volume should not

Total internal reflection gives

Long path length High S / N

• Allows for the best

Spectra as extra information for analyte identification

• A solvent with UV cutoff higher than the working

Beer’s law A = log (Io/I) = ×l × C

• Column backpressure P = 1000 (LF / r2dp2)

• Increase in viscosity → decrease column efficiency

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