c


















 



 



 



 


 
 
 !






"#

 
$

%
: Take the best possible measure for the separation power the GC system in use offers. The best
possible separation power measure is the ³TRENNZAHL ( TZ) ´ - see below - and check if the physical
conditions are optimal: that is GAS FLOW measured as dead time tm and TEMPERATURE or temperatur
PROGRAM of the separation system in use.
&'%: The raw retention time tms = ts + tm which is the sum of the residence ime in the tationary
phase plus the residence ime in the obile phase resulting in the total retention times.

%
: Use the best possible measure for the selectivity the separation system in use offers under the
physical conditions taken. This is the retenti on index I difference of two specifically selected test
substances.

There is a simple correlation between the separation power measured as TZ and the difference between
the retention indices of the two substances (a) and (b) to be separated in order to avoid the reason for
qualitative errors: OVERLAPPING of peak (a) with peak (b). There is always a most critical pair of
substances to be separated, therefore these two test substances will do it. They may overlap by 100%.

For the measurement of the separation power ( TZ), the dead time ( tm) as value for the gas flow speed,
the retention index Ia and Ib of the two substances, which sh ould not overlap and if the temperature take
is OK, we need test substances: a system series of consecutive homologues and the substances (a) and
(b). The simplest system test substance series is a mix of n -alkanes, for instance n-hexane, n-heptane
n-octance, n-nonane, n-decane (or much larger members of the paraffine homologues like
C12, C13, ....C 25, C26... easily available as diesel oil and much higher n -alkanes available as paraffin from
candles). In case the chromatographer works entirely in the fie ld of polar compounds the system series
of homologue saturated esters of saturated fatty acids are applicable.

(
 

 
   

 







   TZ


  

 



% (
%) = number of baseline separated peaks between one pair of system homologues. This
separation power value is valid in isothermal as well as in temperatu re programmed GC and even in
HPLC. This makes TZ fundamentally better applicable than the theoretical plate height HETP or the plate
number n = 5.545 * (tms/b05) 2. ³n´ is valid only for the data pair tms and the correlated b05 value and
only uner strict isocratic conditions. The factor 5.545 is valid only for a strict Gaussian peak shape whic
in practice nearly never exists. Thus basically correct and in all range s applicable is

%)*+,+"-,+-"".++"/+-""-01

tms H(n) = total retention time of the homologue (n) in seconds.

tms H(n-1) = total retention time of the homologue (n -1) in seconds.
b(n) = peak width of homol ogue peak (n) measured in half height in seconds.
b(n-1) = peak width of homologue peak (n -1) measured in half height in seconds. The ³minus 1
in the TZ formula above follows from the fact, that the half areas of two homologues already stay
in the chromatogram range given by TZ.
TZ represents the number of baseline separated peaks. It is valid in isocratic (isothermal, isobari
 non programmed) GC as well as in HPLC. Baseline separated peaks still overlap by about 1%.
The TZ value is valid ONLY in the chro matogram range given by tms of homologues n -1 and n.
% 
%)

 






!
Basically TZ will be at least as large as the length of a separation system in m in case of standard
GC but could easily be as large as five times the capillary length in meter for capillary GC.
The maximum TZ ever found experimentally reached a value of about 80. This means: 80 peaks
are baseline separated between one homologue pair. About ten homologues can be separated in
programmed GC run, that is about 800 peaks may be baseline separated in a single capillary gas
chromatogram. Double dimensional or GC x GC separation systems offer more, see the next GC
symposium 2006 with already the third GC x GC symposium - click on www.richrom.com

TZ data are only correct in case the column / capillary is NOT overloaded by the test homologues
like hydrocarbons, esters, alcohols. All types of errors in making and giving the test solutions
result in too small - falsified - TZ values. The b05 values must correlate with non overloaded
homologue peaks. tms is written ³t´ and b05 is written ³b´ in the figure below:

&'%: The beauty of TZ as separation power value for a column/capillary are the following
characteristics:
a) TZ is a practical number. It means ³number of baseline separated substances´ within the
chromatography range given by the homologues eluted ain t he time window between t1 and t2.
b) TZ allows to optimize all physical conditions of chromatography just for this chromatogram range
which is enclosed by the just mentioned two homologues.
c) TZ and the substance quality value ³retention index´ are strict ly correlated. If one knows the retentio
indices of two substances a and b which must become baseline separated for the best possible quantity
results then TZ is measured with the two homologues enclosing the positions for substances a and b.
The found TZ value must be equal or larger [100/(Ib -Ib) + 1] as given in formula [2] below. If the TZ
measured in this chromatogram range is (a bit) smaller, then the mobile phase flow and / or the
separation temperature is changed until this TZ value reaches a maximu m.
The homologue at t1 above may be n -heptane, then the homologue at t2 is n -octane. The used
homologues must differ by one CH2 group. In this chromatogram range a total of TZ substances can be
baseline separated. TZ values depend on the position of the ho mologues at t2 and t1 thus the separatio
power value given by TZ is not a constant number along the whole chromatogram range, but it can be
maximized as mentioned above but also by improving the sampling conditions. For the TZ over the
whole retention tim e scale see the ³ ABT ´ concept discussed in HPLC.

%2
 is the best possible ³address´ of any substance eluted in GC. This is based on th
series of n-alkane HOMOLOGUE retention times. The standard series of test substances starts with
methane (C 1, index 100) and ends with the normal hydrocarbon C 100, index 10,000, but more practicall
with C40, index 4000. The retention indices of the n -hydrocarbons is independent of the stationary phas
the type, length, diameter, temperature, mode of pressure or temperature programming, or phase flow
speed. The retention index of any n-alkane is ALWAYS 100 times its carbon number. Thus the retention
index of n-heptane is fundamentally always and under any conditions seven hundred.

This is NOT true for any non n -hydrocarbon substances like alcohols, esters, acids or one of th e million
of other substances available for GC separation.
Their retention indices depend on the temperature of the separation system and on further details of th
working mode. Under isothermal conditions the retention index Ia is calculated by logarith mic
interpolation between the index of the normal hydrocarbon eluting prior substance (a) - which is I(n-1)
and the normal hydrocarbon I(n) which elutes after substance (a). In temperature programmed GC the
calculation of the retention indices needs the use of polynomial interpolation, as there is no any linearit
between retention time and index.

The correlation of retention index differences for a baseline separation and of the Trennzahl is simple:
The substance (a) has the retention index I(a). The sub stance (b) has the retention index I(b). These
two substances will just be baseline separated if the column / capillary has the necessary Trennzahl TZ
for this substance pair (a) and (b). This value is called ³TZ necessary´:

%)necessary 3*/44.+5+"-5+
""01

6


:
I(a) = 914; I(b) = 929.
than TZnecessary according to formula [2] = 100/(929 -914) + 1 = 7.7

As in classical ³packed column GC´ the TZ value is often double the column length in meter, the data
example above tells which technique to use. Classical packed columns will fail, TZ necessary is too large for
packed column. It is a good idea for error free analysis to know retention index data of the substances t
be separated and the available TZ value of the co lumn/capillary in question. It is a good concept to
master chromatography ahead of sampling: it saves time and material. The needed small formulas are
easy enough to use them even in daily routine work.

The retention index concept as basis for the optimiza tion of qualitative GC (and HPLC) is of utmost pow
but widely underestimated or - even not anymore known. It boomed in the sixties - seventies of the la
century, however NOTHING has changed in this respect in this century. But there is no any better
concept visible.
The simple example above tells, that the retention index concept helps also to optimize quantitative
analyses - but especially it optimizes the stationary phase selection. Because the retention index
difference of two non hydrocarbon substan ces depends entirely on the stationary phase selectivity. Thu
otimization means FIRST to take the best available stationary phase and THEN to use the best mode of
operation. This is the mobile phase flow speed and the best available mode for programming t he
temperature (and or the pressure) in GC and the mobile phase composition in HPLC.

Optimization costs time, but there are possibilities for auto -optimization. The ³necessary TZ´ value can
be entered prior an optimization series of runs. The used optimiza tion software finds this way the
shortest possible analysis time for the necessary baseline separation of the most critical substance pair
in a given sample.

A short summary in between: Not only TZ depends strongly on the mobile phase flow speed.
The retention indices of some selected test substances are a very good measure of the polarity /
selectivity of stationary phases, as already mentioned. This allows to pre calculate which phase is the
best for a critical substance pair separation. Knowing the ret ention index values for the most critical
substance pair allows to pre calculate the necessary Trennzahl. From this value the chromatographer
knows which type and length of column / capillary he needs for a successful separation. With other
words: Retention index and Trennzahl allow to master chromatography. The theoretical plate number
concept - main aspects of the classical theory - is useless in any programmed chromatography. The mo
weak fact of the classical theory is its single substance basis. As ch romatography is a separation
technique, all concepts based on a single substance data are nearly useless in practice. In this site
therefore quite some classical theoretical concepts which are based on single substance values are not
supported.

o

 
 introduced into gas chromatography by E. Kovats and repeated:

The complete series of the normal alkanes is the substance fundament and ranges from methane CH 4 ,
index = 1 x 100 = 100 practically to C 50H102, index = 50 x 100 = 5000. In extreme cases higher norma
hydrocarbons than C 50H102 are applicable as GC ends only near C 100H202 .
Thus n-heptane (C 7H16) has by definition the retention index 700 and n -hexane (C 6H14) has by definition
the index 600. Homologues with carbon numbers differ ing by one therefore have retention index
differences of 100 units. The retention index of these n -alkane test substances is independent of all
variables in GC as they have a fixed index value by definition. All GC laboratories have an easy access t
n-alkanes which are the main compounds in diesel oil. This is basis of the global usefulness of the
retention index concept. Retention index data can - in limits - be pre calculated if the structure formula
of a substance is given.

It is important to know, that the retention index of nearly all non - n-alkanes depend on nearly all
variables a chromatographic system can have, thus identification errors - systematic qualitative errors
can be found with the help of accurate retention index measurements, see under ³Qual. Error HPLC´ main
because the index concept is applicable in HPLC as well.
The fact, that the length of a separation systems correlates nearly linear with the Trennzahl and the
analysis time correlates nearly linear with the separation system length, we have no big chance to reach
with a single column / capillary system a very high Trennzahl. This is even more critical in PLC. There th
total Trennzahl of a one direction separation is limited to 50 (versus 800 in GC and about 100 in HPLC)
Thus we need for multi component analysis a better concept than offered by single dimensional
chromatography. The way out is two dimensional PLC (TZ total around 400), two up to multi dimension
HPLC, chromatography system combination like GC x GC, GC x HPLC, GC x PLC, HPLC x PLC, HPLC X
HPLC. Meanwhile the GC x GC concept reached a high level of practical applicability. The third
international symposium on GC x GC is scheduled for May 2006 in Riv a del Garda (It) -
see www.richrom.com
lComprehensive GC is a next name for GC x GC and was the main topic of the international series on
capillary chromatography symposia already in 2003 in Las Vegas, USA.






"#


$

When is the polarity wrong ?
It is wrong, if the critical pair of two substances ³a´ and ³b´ which we MUST quantitize and therefore fir
separate from each other, have the same retention index. They have the same retenti on time and the
same peak width in half height and therefore the corresponding ³Trennnzahl necessary´ is unlimited
large - see the formula [1] above, which would mean an unlimited long separation system. Even GC x
GC or any other mode of comprehensive chro matography cannot solve the problem physically. We MUS
find a stationary phase with a selectivity making the retention indices Ia differing from Ib, the more the
better.
There are some technical limits to change mechanically columns / capillaries often un til an acceptable
selectivity is found.
There are better ways: IfC found very early the physical change of selectivity / polarity in GC and HPLC
without replacing columns/capillaries.
The concept is very simple and therefore was for a long time not accept ed by classical theoreticians:
Chromatography is TIME based. We connected two chemically differing stationary phases P with N in
series and changed the residence time of the substances in the two series connected phases. P means
³polar´, N means ³non polar ´. The residence time in GC changes either with the temperature and / or
with the mobile phase flow speed. Both effects can be used simultaneously: warmer and faster makes
the residence time of a substance shorter. A cooler phase P and a warmer phase N mak es the total
system polar. A higher mobile phase flow speed in N and a lower in P also makes the total system polar
If now the critical substance pair differs in polarity, the residence time changes will allow for a
separation. The polarity / selectivity change of flow speed and / or temperature in P and N series
connected columns/capillaries can be managed fully electronically, that is completely software driven.
As in practice there are more multi compound mixtures to be analyzed than analytical duties b ased on
critical pair of two substances, the auto optimization is controlled by the total number of separated
substances. The larger this number, the less is remaining overlapping. Finally we may have reached a
selectivity optimum but still peaks may ove rlap. In this case we need the column switching concept
introduced by David DEANS, the ³Deans switching´ technique, see figure 1 below).

Even process capillary gas chromatographs use micro DEANS switching concepts in order to separate
critical substance pairs.

6


 

G = gas inlet, pressure controlled

S = sample inlet
A = non polar column / capillary A
B = polar column / capillary B
DA = detector connected to column A
with a flow resistor capillary of equal
flow resistance as column / capillary B
r = flow resistance, normaly a flat capillary
DB = detector, connected with column B
o,c = open or closed gas flow valves.

,

The carrier gas flows via sample in
through column A. The gas flow through column B
does not pass the sample inlet. Sample injection
starts, the substances are separated through colum
A. In the second the ´critical´ substance pair reach
the end of column / capillary A both carrier gas
switches change the open/close status. The
³critical³ pairs are pushed into column B where a
separation starts. See right side of the figure 1.

A few seconds later the carrier gas switches are set back to the condition at start. Thus remaining
substances in column A continue the separation. Meanwhile the ³critical´ pair is separated in column B
which will surely happen if both substances have truly a differing polarity. If NOT, no separation of
substance a from b is possible and only an excessive Trennzahl will solve the problem. The technical TZ
limit is near 80 as long as no excessively narrow super separation capillaries are applicable.
The DEANS switching concept is now improved by the use of ³micro in line detectors´ which work inside
the separation capillary - let say a few millimeters prior the end of column / capillary A. This way the ga
switching can be started well in time the ³critical´ pair arrives. Modern micro GC is developing very fast

%
 
 there is nevertheless a more promising way to change the separation selectivity - the colum
/ capillary polarity: these are instruments with two differing columns / capillaries connected in series. A
chromatography is based on time, the selectivity can be changed by changing the residence time in one
of two columns / capillaries. This is possible just by physical means:

" by selective changes of the carrier gas flow speed - faster in A, slower in B
" by selective change of the column / capillary tempe rature - warmer in A, colder in B.

%
): The mobile gas phase flow speed is made larger in column A and smaller in column B by adding
or releasing gas at the connection point thus changing the polarity from a mean effect A + B to a larger
or smaller A- or B-effect. Flow speed changes are easily done electronically with very high precision.
Such a chromatography system allows for computer controlled auto optimization for the best possible
separation of substance a from substance b.
%
): The selective change of the temperature for column A versus column B does the same: A warme
stationary phase A reduces the A - polarity and a lower B - temperature enlarges the B - polarity and vice
versa. If the polarity of substance a differs from substance b we will get t hem separated, again under
auto- optimized conditions, as changing the flow speed and/or the temperature is easily controlled by
software. It is the residence time in the stationary phase(s), which controls chromatography by
selectivity changes of a column tandem. If however the polarity and molecular shape of the critical
substance pairs are equal, we succeed only with larger TZ numbers.

There is a further and even more powerful mode of electronically controlled and switchable column /
capillary selectivity. The peak cutting concept of DEANS is combined with the just mentioned electronic
selectivity change of IfC, thus combining both concepts. As the correct quantitation of critical substance
pairs is more often very important, this GC modificatio n is the utmost we can do for reduced quantitativ
systematic errors in GC.

%

 


 
 
 ! 

!2
 

 





 
!2
 

 






!

%
!
Retention indices are the best quality values in gas chromatography and no any other concept - even n
the best adjusted retention time concept - is more useful in practice.
The following conditions however must exist:
a) The temperature must be kept at + - 0.05 degr centigrade or better. The mobile phase pressure mus
stay constant within a few millibars (possible, no problem technically with proper hardware quality), the
fluctuations of the laboratory air pressure must be excluded from impact by the use of an elevated inlet
outlet carrier gas pressure (using constant gas flow resistors) and the peak amount does not overload
the sorption capacity of the stationary phase. With other words: no oversampling.
b) The integration software and the signal producing hardware are so qualified, that the peak retention
time is measured based on quartz clock precision and is polynomial interpolated by fifth degree around
the top of each peak to a precision of + - 0.001 seconds (is possible without problems. Qualified
chromatography instrument companies used this technique already in the last century).

Under those conditions the retention index values reach a precision of + - 0.01 unit. Such a precision
allows very sharp and safe identific ations in case n-alkanes exist in the sample for qualitative GC or are
mixed to the sample. The added alkanes must be kept in such a low concentration, that there is no
impact on the overall polarity / selectivity. How errors in this respect act is shown i n line chromatogram
LOG/LOG
Even a much weaker retention index precision than + - 0.01 unit is often good enough in practice.

NOTE: columns / capillaries having a (quite good) separation power of Trennzahl TZ at 33 can just only
baseline separate two substances with a retention index difference of 3 units.
Now we need the formula for isothermal index calculations to see, where is the danger of serious
systematic errors:
Isothermal GC retention index according to E.Kovats in a formula IfC prefers:
2
5 
 !7*+5o-58"90
+7.o".
+8.o"1/5801

for isocratic separation only !

IB = Index of the n-alkane eluted before substance X
IA = Index of the n-alkane eluted after substance X
tsX = the net retention time of substance X in the stationary phase.
tsA, tsB = the net retention times of the two n -alkanes given above.

NOTE: If no any programming is used, the series of n -alkane homologues elute very precisely in
logarithmic shape. Under programmed conditions - by flow or temperature programming - the
homologue series elutes nearly in a linear shape but NOT precisely linear enough Homologue series of
many other substance classes behave under isothermal conditions similar like the saturated normal
hydrocarbons.



The n-alkane eluting before substance X = n -hexane, C 6H14; IB by definition = 600
The n-alkane eluting after substance X = n -heptane, C 7H16; IA by definition = 700
Only the non adjusted (raw) retention time values tms can be measured,   
 



: +
"- 

 . As tms = tm + ts which is ts = tms - tm
it follows, that we must find a way either to measure or to calculate the dead time tm.
In the following example we use capillary GC data and work fi rst with a wrong dead time of tm = 122.3
seconds and next with a correct dead time of tm = 119.55 seconds. The measured non adjusted
retention time values of the two n -alkanes and the substance X are correct. tms -data are not influence
by wrong or correct dead time values, but the needed adjusted (net) retention time values ts are wrong
if tm is wrong. In both tables the following data are used:

tmsB = 450.066 seconds; tmsX = 455.876 seconds; tmsA = 512.754 seconds

A dead time error of 3 seconds, which is a relative time error of only 0.7 %, results in an retention inde
error of 5 full units. Remember the possible qualification of index data: 0.1 to 0.01 units is possible.

tm wrong 


tm: 122.38 119.55

tms B 450.07 ts B = 327.67 330.52

tms X 455.88 ts X = 333.50 336.33

tms A 512.75 ts A = 390.37 393.20

57* ;4<!= ;4!4

IX wrong 57


Using the same raw retention time data as above but the correct dead time value and now with a
tms-error of only 0.1 seconds for the substance X, we find a retention index error of 0.2 units in the da
range given above.


 
:



>

 



 
 
 







! Overlapping peaks which shift the retention time values drastically make
correct qualitative analyses - which also means: correct identifications - impossible. In such cases two
and multi dimensional GC with high resolution capillaries is a must - and available. Total peak
overlapping disables correct qualitative as well as quantitative analysis. The same is true for over
 sampled peaks. Their retention time is falsified, most often shifted to too large ts -values.

The power of the retention index as qual ity scale is the fact, that data tables for automatic identification
are stable in mass analyses for many months, whilst other time based identification values like the
relative adjusted retention time values
tsX / ts(reference) are easily unstable and th e relative values of non adjusted relative retention values tmsX
tms(reference) simply are useless. They even change with the outside weather conditions.

%
!
Retention indices in temperature programmed gas chromatography are correct only, if one leaves the
classical definition given by the inventors of the index. In a longer discussion Prof. Kovats accepted, tha
correct TGC indices need polynomial interpolatio n mathematics, as there is no real linear correlation of
index over the carbon number of the reference series of homologues. All index values must be non
linearly interpolated even between two consecutive n -alkane homologues enclosing the substance X.
Temperature programmed retention index calculations have the following formula:

5  
7*
/97/97 /97/97 </97?0<1

a, b, c, d, e, f are constant polynom factors found for the n -alkane homologue series of seven memb er
which at best are mixed to the sample. This guarantees, that all physical conditions of programmed
temperature with the additional effect of changed mobile phase flow during the analytical run are kept
equal.
For the polynom analysis see ³ Calibration´ .

Conclusion: identification by GC should be done isothermally. Backflush and cut techniques allow to do
this with such a big success, that the application of MS and MS/MS for id entification purposes only are
often not necessary. The precision retention index and the use of high resolving separation systems doe
it. The situation however is different if structure elucidation is the task. Then capillary GC, at best GCxG
online a detector pair FID parallel to ECD or ECD parallel to MS/MS is necessary.
ECD = erlectron capture detector, highly non linear with a very narrow working range; FID flame
ionization detector, S-shaped non linear but with a wide working range. ECD and FID are quite sensitiv
detectors.

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