HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY
MUHAMMAD TAUFIQ BIN ROSMAN 55105118014
NUR FARHANAH BINTI RADZMAN 55101118017
NIK NUR NATASHA BINTI ZAINAL ABIDIN 55103118003
WAN NUR PUTRI LIYANA SHAH BINTI 55104118028
WAN ABDUL HALIM
 HPLC OPTIMISATION
EFFECT OF
TEMPERATURE

 In many critical HPLC analyses , variations in temperature can cause significant

changes in retention times.
 this making qualitative analysis difficult and affecting the precision of quantitative
measurements.
 Elevated temperatures are advantageous because decreased mobile-phase
viscosity, increased mass transfer and increase sample solubility result in either
better resolution or faster analysis
 For example the same materials are separated at different temperatures while the
mobile-phase flowrate and composition are held constant
 SENSITIVITY
• Detector sensitivity, sensitivity of the detector is a measure of its ability to
discriminate between small difference in analyte concentration.
• It is actually the slope of the calibration curve.
• It is also dependent on the standard deviation of measurements.
• The higher the slope of calibration curve the higher sensitivity of detector
for that particular component.
• Sensitivity of a detector is not the minimum amount that can be detected.
• This value is influenced by the chromatographic conditions.
 ACCURACY
• Accuracy can be defined as the amount of uncertainty in a measurement
with respect to an absolute standard. Accuracy specifications usually
contain the effect of errors due to gain and offset parameters.
• To get the best accuracy of readings is by making sure that the mean
recovery will be 100+2% at each concentration over the range of 80-120%
of the target concentration.
 Increase and
Lower separation
decrease retention
efficiencies than GC.
time.

LIMITATION

Lack of sensitive and

Need more
specific detectors in
experienced
application involving
operator
complex sample.
SAMPLE PREPARATION

If the sample in mobile

Prepare Standard and
phase, calculate the
Sample Solutions from
amount of mobile
the same solvent.
phase (M1V1=M2V2)
 ADVANTAGES DISADVANTAGES
 Offers a highly accurate  Can be costly,requiring large
method to identify certain quantities of expensive
chemical components in a organics.
sample.

 Higher resolution and speed  Complexity

of analysis

 Can be reused without  Intensive sample preparation

repacking or regeneration
 INDUSTRIAL APPLICATION

Pharmaceutical Industry Clinical Diagnosis