Quantitative Micro Planar Chromatography µ-PLC file1 Quantitative Micro Planar Chromatography...
Transcript of Quantitative Micro Planar Chromatography µ-PLC file1 Quantitative Micro Planar Chromatography...
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Quantitative
Micro
Planar Chromatography
µ-PLC
240908
Drs. Olga & Rudolf E. Kaiser,
Institute f. Chromatography
P.O.B. 1141, D-67098 Bad Duerkheim, Germany
www.interchromforum.com
www.institut-f-chromatographie.de
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Circular is the mother mode of
PLC
• Because everything ‚runs‘ circular.
• And the circular procedure is the better
and the easier way. In all aspects.
• There was however a fundamental
problem: circular chromatograms could
not or not simply be quantitized.
• But this is history.
• Therefore µ-PLC could now be
developed.
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µ-PLC
µ-PLC differs fundamentally from TLC,
HPTLC, OPLC but is similar to HPPLC.
It is circular planar chromatography.
The stationary phase layer lies
horizontally flat.
There is no mobile phase chamber, but
a thick glass plate covering the plate
layer in 1 mm distance. It causes a
virtual vapour filled chamber.
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100 x 100 mm HPTLC plate
5 mm thick glass cover
plate with central hole
Position fixing adhesive
stripe, it holds also
analysis codes
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A manual analytical method ? Still today ?
YES, because:
* Often there are only a few products to be compared.
* The comparison is absolutely safe if the samples are
taken and given perfectly.
* We have full PLANARITY. It is at least two dimensional
and no any uncertainty by polarity gradients, tempe-
rature, flow differences can exist in the ‚overlapped‘
chromatography area – therefore FEW samples !
* The compare run is strictly simultaneous.
* Take THREE equal authentic samples and three
questioned samples on one plate for utmost security.
* Even with one sample security is given as we can
include inner standards along the single circle lines.
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PHASES:
0.5 to 1 ml from a closed flask
through a wick onto the thin
layer from upside to down.
No pump or pressure used.
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precise centre position is kept by
contacting three aluminium rods
a, b, c
a
b
c
µ-PLC needs centre precision prior separation
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SAMPLING:
Samples from GC capillaries, from
HPLC column outlets or normal
sample solutions are given directly
or by filled micro capillaries
or by µ-brushes.
The samples are spotted
next to the plate centre.
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Good sampling precision is possible
by the tools shown below but denied by
experts until they really check it….
Micro brushes work from a few to many microliters and
remain clean even with non filtered samples….
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Sampling and
focusing by
water colour
brushes
nr. 00 - 6
Brush nr volume sampled repeatability spot size (mm) contact
00 2.7 micro liter + - 6 % 4.5 +- 0.26 10 sec
1 4.7 5 5.9 +- 0.3 10
3 8.1 4 7.8 +- 0.2 15
5 17.1 4 11.3 +- 0.4 15
6 33.7 1.6 15.9 +- 0.2 20
quite precise,
easy to use.
50 ct/pc
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No problem: bad sample positions are
corrected by circular focusing :
sampled
focused
separation
started
However the final focusing
position MUST be accurately central
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Sampling (here by brush no.1)
3 x spotted
2 x spotted
single spot
Position fixin
gtape, ho
lds
analytica
linfo
Keeping position precision is necessary in case of
resampling and/or phase changes
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• Sampling in circular PLC is
fundamentally more
accurate than in standard
TLC / HPTLC.
• However we need some pre & after
treatments:
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Important SAMPLE
treatments
• 1.1 total COLD removal of
sample solvent(s)
• 1.2 FOCUSING , positioning
and removal of the
focusing liquid.
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Manual sampling of 2 to 8
samples using
artists water colour smallest
micro brushes or fused silica
capillaries….
No problem.
Just some minutes training and one
realizes the analytical benefits.
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or 1 full ml sample to the plate; dry and
Enriching here
Steadily flowing gas
(dry air, CO
2
, N
2
…)
1 ml
If necessary: + 50 degr
by a heated plate under.
One to two extra holes
In the glass cover plate.
focus
to one
sharp
circle
The virtual 1 mm
gas chamber is
open to all sides
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The one ml „mass volume“ sampling
needs a constant gas flush by
2 L / min air, nitrogen or carbon dioxid.
The use of simple gas pump
does it.
NOTE: it offers ppt-analysis (100 -->1 ml, ng)
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The gas pump provides an easy and
effective use of the chromatography
extra power based on gas phase over
and within the stationary phase
Gas Phase Problem:
– not at all used by the PLC
community. Most helpful with
HCOOH, NH
3
, C
2
H
5
OH traces & more
specifically acting vapor traces.
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DEVELOPMENT
• 3.1 ONE PHASE. 500 to 750 µl per run
given from a one ml flask – isothermally.
• 3.2 differing liquid PHASES (MMD)
wet-in-wet or by in between drying.
• 3.3 SIMULTANEOUSLY 3 to 4 PHASES:
used only for a separation optimization.
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Plate cleaning first…
then
dry
dry, sample, dry again
focus the sample(s), dry again, separate, look,
make the photo, take the data chip, quantitize,
report, use the results for a decision….
Standard procedure:
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The µ-PLC chamber is virtual,
the mobile phase runs under constant flow.
Sample spots are focused to arcs, dried,
separated by a phase from a 1 ml flask….
100 mm
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Separation of 8 samples (0.5 ml phase
in total from a 1 ml flask will do it.)
Economy with
expensive
phases:
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µ-PLC -
QUANTITATION
By digital photography only,
as the classical scanners are
too slow,
they kill separation power &
fail in circular chromatography.
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Quantitative µ-PLC data could be
reproduced by
+- 0.05 % relative, ng-level
because the concept reduces the plate structure
(this is comparability standard deviation
with N between 4 and 12; standard is +- 0.1 to 0.5 %)
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1. Is too dark. Now
2. Light is improved.
3. Select integr.arc.
4. Make light uniform
1
2
3
Improvement of digital photos. Example:
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• Circular quantitation software written
by SORBFIL experts, based on IfC ideas:
• Drastic reduction of the plate surface
structure signals by
N => 4 track quantitations. Each track
at a different angle within one µ-PLC single
sample bow or a circle part.
• Factor of ten higher precision than by
scanner quantitation – expressed as
comparability standard deviation.
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Load chrom.picture into SORBFIL 2.0, select arc,
select number of tracks(=4), angle(=4), start and front.
Quantitation by SORBFIL v. 2.0
example:
red=centre
green=Rf 1
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Analogous data: 7 peaks/track
NOTE: all analog signals of 4 tracks cover each other
1
2
3
4
5
6
7
peak
peak
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and digital values, 7 substances, N=5
Nr area RSD% height RSD%
1 11154.8 0.937 2571.2 0.718
2 11003.8 0.525 1807.0 0.639
3 38429.8 0.600 4596.8 0.272
4 46302.4 0.350 4000.0 0.163
5 39413.0 0.520 3502.0 0.093
6 7733.8 0.695 978.8 0.606
7 24617.0 0.143 2052.8 0.156
S 178645 0.164 % 19509 0.091 %
RSD = 100 x rel. standard deviation / mean
This data quality is possible because of plate structure reduction
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Analog signal
showes the
resolution
(Trennzahl) of
a FLU
chromatogram.
It reaches here
the separation
efficiency of a
short HPLC
column
(TZ=50)
Fluorescing product, two main substances:
two traces
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Mobile phase optimization in PLC is
time consuming.
The circular technique allows to separate
with three or more differing mobile
phases simultaneously.
ONLY circular PLC allows this elegant
procedure.
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µ-PLC is NOT regulated
It is a free analytical mode offering a very
high level and savety of correct decisionmaking for
the increasingly important question:
• Is substance or mixture A really equal
to substance or mixture Q ?
For the analytical answer we do NOT need
any structure specific detection, just only very well
comparable chromatograms. This is available using the
power of truly PLANAR separations
of samples partially overlapped –
see the application example.
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A
T
Q
Application: MEDICINE COMPARISON
A = „authentic“ med. Q = is it really „authentic“ ?
T = a PLC test mix to check the run conditions.
If A NOT Q
then Q is not
the authentic
medicament.
FOR SURE !
All under UV; A touches Q
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A
A
Q1
Q2
T
T
Q
5 to 50 mg into 1 ml ethanol/water
5 mm spot T, A and Q solution.
Dry, focus, dry, start with weak
mobile phase, if necessary go
ahead with stronger phases.
Photo, quantitize (if necessary).
3 to 4
pills :
A is
NOT Q,
NOT Q1,
NOT Q2.
T
Q
A
0712281733/tabW/V 0802241829/tabM/H
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4 „equal“
pills:
partially
overlapped
for saver
identification
A = Q2 ?
Q1 is not A
Q3 not A not Q2
All 4 are said to be equal
overlapping
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Limitations
• µ-PLC needs PLC knowledge.
• It needs circular chromatography software,
digital camera and a fast enough laboratory
computer off-line.
• It is limited for only in maximum 24 (or 400?)
samples per plate but 3 - 4 is optimal.
• The smallest plate size is limited to
10x10 mm, standard = 100x100 mm.
• It is made mainly for critical product
comparison.
For this task: Two samples are standard.
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From where to get µ-PLC hardware ?
Definitively NOT from the HPTLC instrument makers as µ-PLC is too economical
It is easy to make your own µ-PLC
equipment .
If there are really problems to get materials and data
just email the author.
From where to get µ-PLC circular software ?
Contact www.sorbfil.com, go to NEWS,
order version 2.0