The document discusses qualitative errors that can occur in high performance liquid chromatography (HPLC). It describes several sources of qualitative errors such as poor separation power, substances being strongly adsorbed to the stationary phase, and changes in stationary phase selectivity from impurities in the mobile phase. It also discusses reasons to use error control techniques in qualitative HPLC, such as checking separation power and using two columns in series. The document introduces the ABT concept for evaluating column quality using retention time and peak width values of separated homologues under isocratic conditions. Plots of ABT data and theoretical plate numbers from an example HPLC column are also presented.

1. QUALITATIVE ERRORS IN HPLC

2.  Poor separation power.
 Some special substances from the samples can
be adsorbed strongly in and on the stationary
QUALITATIVE ERROR SOURCES IN HPLC:
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.

3. 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.

4. 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

5. 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

6. 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.

7.  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.

8.  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 C18 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.

9. 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.

10. 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.

11.  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.

12.  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.

13. 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”

14.  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 = 5.545 * (tms / b05)2 = 5.545 * ((ts + tm) /
(b0 + a*(ts / tm))2
 HETP = L / n [mm]

15. 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.

16. 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).

17. FIGURE 1: ABT DATA FROM THE HPLC COLUMN
MENTIONED ABOVE UNDER CONDITIONS NEAR
THE TZ MAXIMUM.

18. 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)