PREPARED BY VAISHNANI BHAVIN M.PHARMA-I (Q.A.)

Qualitative error sources in HPLC: Poor separation power. Some special substances from the samples can be adsorbed strongly in and on the stationary phase. Selectivity changes of the stationary phase can also result from highly sorptive impurities in the mobile phase. Many mobile phases are oxygen / light sensitive which let grow the concentration of unwanted mobile phase impurities.

Cont… The column temperature Strong believe in the identification power of spectrographical HPLC detectors The total numbers of chemical substances qualified for a chromatographic analysis is very much larger in HPLC.

Minimum of reasons which should animate using error control techniques in qualitative HPLC Check the separation power of the chromatographic equipment. Use of two columns either parallel or in series under conditions of a physical and chemical selectivity optimization Planar chromatography in connection with HPLC

ABT - concept The ABT concept uses retention time and peak width values of homologues separated isocratically. A=a= constant value as column/capillary quality number. B = b0=peak width value T=tm=dead time/residence time

Condition for ABT concept At least four homologues are used. The time and peak width data must be taken very precise and the isocratic conditions must remain constant during the complete run time. As no peak overloading is acceptable - which falsifies peak width and retention time data. The homologue sample is diluted in a solvent eluting later than the homologues.

From this run the retention time and peak width data allow to calculate most accurate and helpful control values. They describe the quality of a column/capillary and the overall qualification of the complete analytical system. The series of runs start at a quite high separation speed, which then is lowered stepwise to a quite low speed. This produces very fast and finally quite slow chromatograms. Analyzing all control data over the speed axis we find the best possible separation capacity at the most economical chromatogram speed.

The best possible stationary and mobile phases at a well selected separation temperature are taken. In HPLC this may be a selection of four consecutive members for homologues from the series of methyl- to C 18 esters of di nitro benzoic acids. ABT data are in easy reach using proper software, but they can also be found by pencil, liner and paper. In HPLC the homologue numbers have independent of the taken ester homologues the index or homologue numbers 100, 200, 300 and 400.

ABT calculates the following basic values  The dead time (tm) or the residence time Knowing the tm value all non adjusted retention time values “tms” can be changed into the adjusted net retention time values “ts”. “ts” is the individual residence time in the stationary phase. Knowing “ts” values we can calculate in isocratic HPLC the important value “k”. “k = ts / tm” or k = (tms - tm) / tm.

Cont… Using k as chromatography unit we can build a chromatography scale in k - units. The chromatographic process - but not yet the separation - starts at k = -1. Any substance or mixture which is insoluble or not sorbing on and in in the stationary phase leaves the instrument at k = 0, also known as “blind time area”. virtual k-value k = unlimited, for substances which are irreversibly adsorb or chemisorb in or on the stationary phase or any solid wall within the instrument.

 The peak width value “b0” b0 is found by a simple mathematical procedure in extrapolating a linear increase of peak width data over tm-time units. bo is depend on sampling process, all mechanical connection parts and their dead volumes, with the mobile phase volume, with the volume of connection tubes and the phase flow speed. b0 is acting completely against separation. peak width value adds to all correct peak width values of all substances in isocratic HPLC.  Because of the strictly linear growth ABT calculates the function peak width = a * k + b0.

All three ABT data: “a”, “b0”, “tm” in seconds change drastically with the mobile phase flow speed “U”. U= tm / L (sec/mm) L= length of the column/capillary (mm). To avoid any trouble caused by not eluted substances with too long retention times every HPLC instrument must have “backflush” installations.

Practical application of “a”, “b0”, “tm”: Any not adjusted retention time value tms can be transferred into k-values. The peak width b05 of any peak with known tms value can be pre calculated: b05 (at tms) = b0 + a * (tms - tm) / tm = b0 + a * k. The Trennzahl for any time region can be pre calculated, see figure 2: TZ = [tms homol.(N) - tms homol.(N-1)] / [b05 homol.(N) + b05 homol.(N-1)] + 1 The peak capacity “PC”

The ABT theory works with the theoretical plate height “h” or “HETP” respective the theoretical plate number n to describe the separation capacity of a column packing or the separation power or packing quality of the column as such. n = * (tms / b05) 2 = * ((ts + tm) / (b0 + a*(ts / tm)) 2 HETP = L / n [mm]

The formula for n and HETP above has the following problem The factor “5.545” is correct only for symmetrical GAUSS shaped peaks. Those do not exist in practice. n and HETP are substance dependent. Separation exists only if there is more than one substance in a sample.

HPLC ABT- and TZ-values  Isocratic runs with a 250 mm long HPLC column of 3 mm inner diameter, stationary phase Eurosphere 100 / C18, mobile phase methanol / water 85 /15 v/v, room temperature, inlet pressure ranges at 8 levels from 255 bar (fastest flow speed) to 39 bar (longest analysis time).

Figure 1: ABT data from the HPLC column mentioned above under conditions near the TZ maximum.

Figure 2: TZ data from the HPLC column mentioned above. The TZ values as function of the mobile phase flow speed (between 0.3 and 2.0 mm/sec)