Universal analyte detector.
Principle-Change of refractive index of the eluent from the column with respective to pure mobile phase.
Very temperature sensitive
FD – Fluorescence
Principle-Enable fluorescent compounds present in mobile phase to be detected by passing the column eluent through a cell irradiated with ultraviolet light and measuring any resultant fluorescent radiation.
Very sensitive and selective
Normally refers to amprometric or coulometric detectors
Principle-Measures the current associated with the oxidation or reduction of solutes.
The detector usually contains low volume cell through which the mobile
phase passes carrying the sample components.
18.104.22.168 Fundamental parameters of HPLC (Fig 1 F6)
Fig 1.6 Fundamental parameters of chromatography
Where, w1/2 = peak width at half height
w= band width of the peak (intersection point of the inflection tangents with
the zero line)
A = peak front at 5% of peak height to peak maximum
B = peak maximum to peak end at 5% of retention times
t0= dead time of a column (retention time of un retarded substance)
tR1, tR2 = retention time of components 1, 2.
t?R1, t?R2= net retention time of components 1, 2.
Retention time (tR):
It is the time between the samples injected and chromatographic peak recorded. The total retention time (tR1 or tR2) is the time, which is needed by sample component to migrate from column inlet (sample injection) to the column end (detector). The net retention time (t?R1 or t?R2) is the difference between total retention time and dead time i.e. the time the sample component remains in the stationary phase.
It is a measure of quality of separation of adjacent bands in a chromatogram; obviously overlapping bands have small Rs values. It is calculated from the width and retention time of two adjacent peaks.
Ideally RS should be greater than 2.
R_s= (2 (t_2-t_1))/(w_1+ w_2 )
t1 and t2 are the retention time of the first and second adjacent bands.
W1 and W2 are their baseline bandwidths.
Reliability of calculation is poor if Rs is 2.0
Repeatability RSD ? 2 % for N ? 5 is desirable
Relative retention Not essential as long as the resolution is stated.
Resolution (RS) RS > 2 between the peak of interest and the closest eluting interfering peak.
Asymetry factor Not less than or equal to 2 is desirable
Theoretical Plates In general should be > 2000
22.214.171.124 Quantitative analysis in HPLC
Three methods are generally used for quantitative analysis. They are the external standard method, the internal standard method and the standard addition method.
1. External standard method: The external standard method involves the use of a single standard or up to three solutions. The peak area or the height of the sample and the standard use are compared directly. One can also use the slope of the calibration curve based on standard that contain known concentrations of the com