HPLC Aruna
HPLC Aruna
HPLC Aruna
HPLC
H or HETP = A + B + C υ
υ
where, A represents eddy diffusion
B represents molecular diffusion
C represents rate of mass transfer
υ represents flow rate
Basic Components of an HPLC
System
1.Pump System.Mobile phase pressures up to 6000 psi are necessary to
achieve reasonable column elution times (~ minutes). Typical flow
rates are 0.1 to 10 mL/minute.
2.Injection System.Used to introduce small samples (0.1 to 500 μL) into
the carrier stream under high pressure.
3.Reservoirs (Solvents).Multiple solvents are necessary for performing
gradient elution's (i.e. changing the polarity of the mobile phase
during a run).
4.Chromatographic Column. Typically 10-30 cm in length containing a
packing of 5-10 μm diameter. Many types of columns are available,
depending on the type of liquid chromatography desired.
5.Detector.Many types are available including UV, IR, refractive index,
fluorescence, conductivity, mass spectrometry, and electrochemical.
Diode array detectors are used when wavelength scans are desired.
HPLC Basic Instrumentation
Separation
Mobile Pump
phase
Sample Injection
Data Processor
HPLC system
• Solvent Reservoir
• Degasser
• Injector
• Column &oven
• Detectors
• Recorder (Data Collection)
PUMP SYSTEM
HPLC Pump
• Solvent Reservoir
– Usually one or more glass or stainless steel reservoirs each
of which contains 200-1000 ml of solvent
• Isocratic elution - single solvent separation technique
• Gradient elution - 2 or more solvents, varied during
separation
SAMPLE INJECTION SYSTEM
HPLC Autosampler and Injector
from Pump to Column from Pump to Column
Needle
Vial
LOAD INJECT
Measuring Pump
HPLC Column
HPLC Detector
UV/Visible Spectrophotometer
Column
– straight, 15 to 150
cm in length; 2 to 3
mm i.d.
– packing - silica gel,
alumina, Celite
CHARACTERISTICS OD
DETECTORS
Have high sensitivity and the same predictable response
Respond to all solutes, or else have predictable specificity
Have a wide range of linearity
Be unaffected by changes in temperature and mobile-phase
flow
Respond independently of the mobile phase
Not contribute to extra column band broadening
Be reliable and convenient to use
Have a response that increases linearly with the amount of
solute
Be nondestructive of the solute
Provide qualitative information on the detected peak
Have a fast response
Ultraviolet detector cell for
HPLC
• Problems caused by dissolved air(O2, N2)in mobile phase
– Unstable delivery in pump
Degasser
– Bigger noise and large baseline-drift in detector cell
Ready
inject
Rheodyne
Injector
through pump
Column
through C
pulse
dampener
A B
from solvent to
Ternary Pump reservoir detector
Chromatography II: HPLC
HPLC Waste Collection
– Use a 10 mL volumetric pipet to add 10.00 mL of soft
drink to 10.00 mL of deionized water.
– Mix thoroughly and half fill a HPLC vial with
your sample. Label the vial with your name and the
name of the soft drink.
• Analysis of Results
– Use the four caffeine standards to prepare a
calibration curve (graph) that plots peak area vs.
concentration.
– Draw a “best fit” straight line on your graph.
– Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.
– Use the concentration of caffeine in your sample,
along with the dilution equation, to determine the
concentration of caffeine in the soft drink you used.
HPLC Chromatogram of Standard 1
M1V1
M2 =
V2