Quantitative Micro Planar Chromatography µ-PLC file1 Quantitative Micro Planar Chromatography µ-PLC 240908 Drs. Olga & Rudolf E. Kaiser, Institute f. Chromatography P.O.B. 1141,

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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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µ-PL-Chromatograms, made by µ-PLC

FLU

VIS

UV

Substances and detection modes differs.

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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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virtual µ-PLC chamber by 1 mm distance

glass parts at the four corners

mobile phase

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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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1 ml mobile phase bottle

wick

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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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Focusing by CH

3

OH, then drying by gas

2 L air, CO

2

, or N

2

/ min

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Important µ-PLC tool :

Gas pump

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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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VIS

Light change from

white through

RGB 100-100-100

to many others

like RGB 0-0-100

UV

FLU

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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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Integrate, see analog data to check…

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4 tracks, 1 degree change

A quantitative example with uniform arcs:

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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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enhanced separation efficiency

4 tracks, angle > 1 degr

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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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Three phases simultaneously

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Four phases simultaneously

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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

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Thank you

Baikal lake in east syberia by Dr.Olga Kaiser