T-Squared Technology, Inc

903 Sneath Lane, Suite 125, San Bruno, Ca 940, ! S "

Tel# 50-$%1-059 &a'# 50-$%3-49
in(o# )))*t+qtech*co, e-,ail# in(o-t+qtech*co,
Chro,atotron
.
$924
Operation Manual
TM


WARRANTY
The Chromatotron is warranted for a period of one year from date of delivery
to be free from defects in workmanship and materials. All defects appearing
more than one year from date of delivery shall be deemed to be due to normal
wear and tear. ince the e!uipment and information given may be applied
under conditions beyond our control" the user must determine the safety of
procedures described and materials supplied" under the user#s conditions.
The warranty does not cover breakage of glass parts" corrosion from any
cause or damage to anodi$ed surfaces.
R%T&RN '()*CY
*f there is a problem with a Chromatotron or parts" submit a description via the
Other Requests/Comments/Technical Questions form in the chromatotron.com
website. *f it is necessary to return items" a +erchandise Return Authori$ation
may be re!uired. ,o not return items without authori$ation.
TRA,%+AR-
Chromatotron" .arrison
Teflon" %. *. ,upont
+ineralight" &/'
A0a1" Colgate 'almolive
2342.44.35
CA-T*ON
6 The Chromatotron is to be used only by those trained in the use of
laboratory e$uipment and solvents"
6 /lug into a 7 pin 8grounded9 eletrial outlet only"
6 +o not leave the Chromatotron unattended when solvent is flowing"
6 -nplug the Chromatotron if for any reason the rotor does not move
freely"
6 The Chromatotron must be used with nitrogen or other inert gas"
6 The Chromatotron should be used in a hood with a good air flow to !eep
solvent vapor away from the operator"
6 ,eep organi solvents away from the motor and eletrial system"
6 After unplugging the Chromatotron% allow the rotor to ome to rest without
applying any bra!ing fore" (1ternal bra!ing may loosen the rotor and
damage the lid"
6 +o not use ra!ed rotors"
6 Organi solvents must be allowed to evaporate co,/letely from sorbent
layers before further drying by heating" Rotors that have been used with
solvents 8espeially methanol9 must not be plaed in an oven"
6 #ilione oils and greases should not be used on or near the Chromatotron"
The rotors may be ontaminated irreversibly"
6 #ilia gel and other finely powdered materials should be handled in a hood"
6 Wear suitable eye protetion"
6 -se a filter to protet the pump from abrasive impurities in samples"
6 /umps re$uires solvent for lubriation" +o not leave pumps running without
solvent for more than a few min"
6 +o not pump to1i samples without suitable preautions to allow for
aidental e0etion under pressure"
::::::::::::::::::::::::::::::::::C01T21TS
/age
TH( CHROMATOTRON AN+ HOW *T WOR,#
*ntrodution" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 5
#(TT*N) -/ AN+ -#*N) TH( CHROMATOTRON
*nstallation" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ;
The Main .essel" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ;
The Teflon <id" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ;
The #olvent *nlet" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " =
#olvent /umps" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " >
Nitrogen 2low" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 4
Changing Rotors" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ?
#orbent <ayer Thi!ness" " " " " " " " " " " " " " " " " " " " " " " " " " " ?
/repurifiation of the #ample" " " " " " " " " " " " " " " " " " " " " " " " 53
#olvent Choie" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 53
#olvent Addition" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 55
*ntroduing and (luting the #ample
*ntroduing the #ample" " " " " " " " " " " " " " " " " " " " " " " " " " 5@
*ntroduing <ess #oluble #amples" " " " " " " " " " " " " " " " " " 57
Ma1imum and Minimum 2low Rates" " " " " " " " " " " " " " " " 5;
Rapid Chromatography" " " " " " " " " " " " " " " " " " " " " " " " " " 5;
*nterrupting #olvent 2low" " " " " " " " " " " " " " " " " " " " " " " " " 5=
Heavy <oading" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 5=
<ight <oading" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 5=
+etetion of -. Absorbing Compounds on the Rotor" " " " " " 5=
+etetion of Colorless -. Transparent Compounds" " " " " " " 5>
2ration Colletion" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 5>
CleanAup and Regeneration of the #orbent <ayer" " " " " " " " " 5B
Multiple +evelopment" " " " " " " " " " " " " " " " " " " " " " " " " " " " " 54
Reyle" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 54
Conneting Chromatotrons in #eries" " " " " " " " " " " " " " " " " " 5?
COAT*N) ROTOR# W*TH #ORC(NT#" " " " " " " " " " " " " " " " "
*ntrodution" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " @3
#orbents% Cinders and /hosphors" " " " " " " " " " " " " " " " " " " " @3
Reipes" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " @5
Coating Rotors -sing Reipes 5A=% )ypsumACound <ayers
*ntrodution" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " @;
Cleaning and setting up the Rotor for Coating" " " " " " " " " @=
Mi1ing and /ouring the Reipe" " " " " " " " " " " " " " " " " " " " @=
+rying the #orbent <ayer" " " " " " " " " " " " " " " " " " " " " " " " " @>
#raping #orbent <ayers" " " " " " " " " " " " " " " " " " " " " " " " " " " @4
#torage of Coated Rotors" " " " " " " " " " " " " " " " " " " " " " " " " " @?
Coating Rotors -sing Reipe >% )lue A Cound <ayers" " " " " @?
/artition Chromatography% Reipe ;" " " " " " " " " " " " " " " " " " 75
Correting Cand #lope" " " " " " " " " " " " " " " " " " " " " " " " " " " " " 7@
/age
MA*NT(NANC( AN+ R(/A*R#
(letrial Connetions" " " " " " " " " " " " " " " " " " " " " " " " " " " " " 77
#olvent /umps" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 77
Ad0usting the Main .essel" " " " " " " " " " " " " " " " " " " " " " " " " " 7;
The Teflon <id A Main .essel #eal" " " " " " " " " " " " " " " " " " " " 7;
Replaing the Rotor #upport Collar" " " " " " " " " " " " " " " " " " " 7;
The 2elt #eal" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 7;
Wear and Tear" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 7;
TRO-C<(#HOOT*N)" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " 7=
CHROMATOTRON /ART# <*#T" " " " " " " " " " " " " " " " " " " " " " ;3
A//(N+*D
The Test Mi1ture" " " " " " " " " " " " " " " " " " " " " " " ;5
*N+(D" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " ;@
::::::T32 C3405"T0T401 "16 307 IT 7048S
I1T406!CTI01
The Chromatotron is a preparative% entrifugally aelerated% radial% thinA layer
hromatograph" The apparently simple onstrution hides a wealth of novel
design details that ensure good resolution and ease of operation" The parts of
the Chromatotron are shown on pages @ and 7"
Chromatography is performed in a thin layer of sorbent " on a rotor B" The
motor & drives the rotor at a onstant speed by a shaft passing through a hole
in the enter of the main vessel 6.
#olutions of samples to be separated are delivered to the rotor via the inlet I
and wi! 9" (lution by solvent forms onentri bands of separated
substanes whih leave the edge of the rotor together with solvent" Channel 2
ollets the eluate and brings it to the output tube :"
Teflon lid C is transparent to -. light% allowing detetion of -. absorbing
bands" (ight retaining lips 3 hold the Teflon lid on the main vessel"
Chromatotrons an be onneted in series by pumping the output of one to the
input of the ne1t" The output of a Chromatotron may be reyled to the input"
Multiple development an be performed"
The Chromatotron was designed for preparative separations on silia gel and
alumina" *t is not useful for hromatography re$uiring ellulose or reversed
phase sorbents"
5
TH( CHROMATOTRON
E
-"#" /atent no" ;57?;=4
@
TM
TH( ROTOR #-//ORT #Y#T(M
THE CHROMATOTRON AND PUMP SET-UP DIAGRAM
3
:::S2TTI1: !; "16 !SI1: T32 C3405"T0T401
I1ST"LL"TI01
#et up the Chromatotron in a fume hood or similar area designed for the safe
handling of organi solvents" /lae the Chromatotron away from hotplates%
steambaths or other soures of heat"
T32 5"I1 <2SS2L
The main vessel is onstruted from FaetalF polymer" After ompletion of
hromatography% wash out solvents that soften or swell aetal 8hloroform%
dihloromethane% aetone% aetonitrile9 with he1ane% other hydroarbons or
ethyl aetate" The solvents ether% ethyl aetate% tetrahydrofuran% dio1ane%
methanol% hydroarbons and arbon tetrahloride are relatively inert towards
aetal" Aeti aid% trimethylamine and ammonia are aeptable as additives
to hromatography solvents" The possibility of solvent damage to plasti parts
an be redued by leaving the nitrogen supply on when the Chromatotron is
not in use"
Do not use mineral acids, formic or other acids stronger than acetic acid.
Chloroform contains hydrochloric acid which will attack the main vessel, metal
parts and your sample. Remove aid by adding alumina or other solid bases
to the hloroform and he! with wet pH paper" Consider using
dihloromethane in plae of hloroform"
Clean the solvent olletion hannel at intervals and lear the output tube with
a straightened paper lip"
T32 T2&L01 LI6
The polyethylene over should be
plaed over the Teflon lid when the
Chromatotron is not in use" When
the Teflon lid is out of the
Chromatotron% plae it on the
inverted polyethylene overG
;
Maintain the Teflon lid lean" <arge partiles of dust on either side of the
Teflon sheet% near the edge or in the orresponding reess in the main vessel%
will ause solvent vapor lea!s" A moist% soft paper towel or loth will remove
dust with a minimum of srathing"
T32 S0L<21T I1L2T
#rew the inlet fully down into the holder in the Teflon lid"
5aintain the )ic= in good condition* " +hort, )orn or >ent )ic= )ill
/a++ +ol?ent a+ a +erie+ o( dro/+ that gi?e an une?en (ront on the rotor*
#traighten the wi! whenever the inlet or Teflon lid is removed from the
Chromatotron"
=
Changing the )ic=
5 /ull out the wi! and wire wi! holder"
@ Replae the wi!% insert into the inlet and
push in with a straightened large si'e
paper lip"
7 Trim the wi! to about 4 mm"
-se the spare wi! supplied or a similar thin
fluffy polyester string" #ewing otton or
thread are not reommended"
S0L<21T ;!5;S
A pump is used not only for solvent addition but also for introdution of the
sample solution and for reyle of eluted omponents ba! to the input"
The pump must have a low dead volume to prevent dilution of samples" /iston
pumps and other pulsating types must also be asynhronous to avoid
synhrony with the Chromatotron rotor" /eristalti pumps are limited to the few
solvents ompatible with fle1ible tubing" The only suitable ommerially
available pump isG
Model RH#YOO#TY<2 from 2luid Metering *n"% www"fmipump"om
/ump onnetions !it% at" no" HA=7 is also re$uired"
>
Model RHSYOOSTYLF from fluid metering Inc.
Pump connections kit H-53 is also required.
&luid Connection+ (or ;u,/ 5odel 4;:150-0SS@
This pump model is no longer available from 2luid Metering% *n"
&luid Connection+ (or ;u,/ 5odel 43S@00ST@L&
B
#rew tube end fittings into the pump gently by hand onlyH
Raise the solvent reservoir @3 m if high ambient temperatures or volatile
solvents redue flow rates by forming vapor lo!s 8bubbles in the pump9"
;u,/+ are +en+iti?e to a>u+e#
6 The otton filter should be in plae at all times when pumping sample
solutions or solvent" #olid impurities must not enter the pump"
Change the otton at intervals" #yntheti material sold as Fosmeti
puffsF is most suitable" +o not use glass woolI abrasive glass partiles
will be released into the pump"
6 +o not leave solutions of ompounds in the pump" The piston may bind
to the ylinder as the solvent evaporates"
6 +o not pump hot or supersaturated solutions" #olids may rystalli'e in
the pump"
6 +o not pump mineral aids" Wash out hloroform 8whih generates aid
on e1posure to air and light9"
6 +o not pump to1i materials without suitable preautions to allow for
aidental e0etion under pressure"
1IT40:21 &L07
A flow of inert gas is essential" Most ompounds will partially o1idi'e when
e1posed to sorbents and air"
The flowmeter is alibrated for air and is reasonably aurate for nitrogen and
argon" *f a bubbler is used% plae it on one side for high flow rates to allow the
gas to pass over rather than through the li$uid" Choose a li$uid of low
volatility% e"g" a phthalate ester" Do not use silicone oil" #iliones may
irreversibly ontaminate the rotors% preventing adhesion of the sorbent layer"
.olatile li$uids suh as 1ylene are unsuitable if -. absorption is used for
detetion of ompounds on the rotor" Do not use sulfuric acid in wash bottles.
Cefore using the Chromatotron% flush out air with &lo),eter Scale
a nitrogen flow of @=3A5333 mlJmin then redue to
about 5= mlJmin 8read at enter of ball9 and maintain
this rate during the hromatography" The latter flow
rate orresponds to about 5 A @ bubblesJse from a >
mm i"d" tube in a bubbler" (1essively high rates will
dislodge sorbent"
Solvent vapor will pour out from the nitrogen inlet if it is
left open to the air.
4
C3"1:I1: 40T04S
<ift the retaining lips with a finger or thumb and slide ba! about 5 m" The
Teflon lid an now be removed" Hold the shaft ollar stationary when srewing
the endplate in or out" Alternatively and more onveniently% turn the shaft ollar
and hold the endplate stationary"
5 -nsrew and remove endplate" @ #lide rotor up with finger 7 Remove rotor"
and plae shoehorn under
lower edge"
> -se endplate in enter hole = #lide rotor up with finger ; <ower new rotor
to slide rotor up into position" to allow removal of shoehorn" into position"
#rew in endplate by hand only"
A tight endplate an be levered loose with the endplate !ey" *nsert into one of
the holes at the side of the endplate" #ee diagram page 7"
*f the sla! between the srew threads on the endplate and the hole in the rotor
auses slight eentriity% loosen the endplate and srew up in a new position
relative to the rotor" Repeat until onentri" (entriity of the sorbent layer%
or the glass rotor% does not affet performane"
S04B21T L"@24 T3IC812SS
1 ,, Layer+" 2or separation of small samples% up to 533 mg per omponent%
@=3 mg total sample"
2 ,, Layer+" 2or larger sample loads% up to 733 mg per omponent% B=3
mg total sample" Also for small samples of low solubility or whih tend to tail"
The resolving powers of 5 and @ mm layers are not signifiantly different for
light loadings"
4 ,, Layer+" 2or very large loads% up to about 5"=g" )ain e1periene with 5
and @ mm layers before using these thi! layers"
<ayers up to a thi!ness of 4 mm an be prepared but are troublesome to use"
?
#raper blades are available for sorbent layers of any thi!ness within the
range 3"7 to ; mm" #ee CHROMATOTRON /ART# <*#T% page ;3"
;42;!4I&IC"TI01 0& T32 S"5;L2
#amples that are free of FbaselineF impurities need not be prepurified" All
samples ontaining very polar impurities should be prepurified by slow filtration
of a solution in a polar solvent% e"g" ethyl aetate% through a layer of sorbent
8olumn hromatography grade9 in a short wide olumn or a sintered glass
funnel" (vaporation of the solvent gives the prepurified sample" This simple
proedure removes very polar ompounds that reat irreversibly with sorbents"
.ery polar impurities will form a line of dar!er impervious spots at the inner
edge of the sorbent on the rotor" -ltimately% bands will strea! and broaden"
Remove the offending sorbent by resraping the layer with a sraper blade
displaed radially outwards from the normal position in the sraping tool"
Removal of @A7 mm of sorbent 8in inrements of l mm% to prevent hipping9
from the inner edge will restore the utility of the layer"
#ubstanes that are rendered insoluble by alium ions are present in rude
e1trats of plant or animal matter" Add gypsum to the sorbent for
prepurifiation of these samples"
S0L<21T C30IC2
The Chromatotron re$uires an Rf lower than for regular T<C" /referably
hoose solvents giving an Rf in the range 3"@ A 3"; using onventional
analytial T<C" A higher Rf is aeptable for easy separations" *f -.
absorption is to be used for detetion% see +etetion of -. Absorbing
Compounds on the Rotor% page 5=% before hoosing a solvent" The range of
usable solvents e1tends from he1ane to methanol" )lueAbound silia gel
layers are not loosened even by a$ueous solvents"
The e$uilibrium onditions in the Chromatotron% versus the non e$uilibrium
onditions of standard T<C% will oasionally ause disparate results" Cands
may separate in the Chromatotron but not on analytial T<C plates% or vie
versa" This effet is most notieable when using mi1tures of solvents with very
different polarities% e"g" hloroform A methanol" The T<C results an usually be
reprodued in suh ases by reduing the proportion of the polar solvent or by
introduing the sample before the rotor is ompletely wetted with solvent 8page
579"
Most ompounds will FtailF when the solvent ontains only low or medium
polarity omponents suh as he1ane and dihloromethane" The addition of a
small $uantity of a polar solvent% e"g" 3"lK of methanol% will sharpen up the
bands onsiderably"
53
<ow solubility of the sample is troublesome with solvents based on mi1tures of
he1ane and a polar solvent% e"g" he1ane A ethyl aetate" *nrease solubility by
replaing part of the he1ane with toluene or dihloromethane while dereasing
the proportion of the polar solvent in order to maintain a reasonably low Rf"
#ee also *ntroduing <ess #oluble #amples% page 57"
Gradient elution is recommended for most separations. Step gradients, from
the bathwise addition of solvent mi1tures with inreasing amounts of a polar
solvent% wor! well" ($uilibration through the vapor spae in the Chromatotron
partially smoothes out the gradient" Increase the polarity much more rapidly
than is usual for column chromatography, using only 3 or steps. A long
smooth polarity hange will give a very large number of dilute frations rather
than an improved separation"
#udden large inreases in solvent density% e"g" a hange from he1ane to
dihloromethane% may ause radial strea!ing of bands" -se a short gradient to
smooth out the hange"
The omplete elution proess should not ta!e more than about 73 min if
solvents giving a reasonable Rf have been hosen"
S0L<21T "66ITI01
2or solvents of normal visosity the usual flow rates areG
5 mm sorbent layersG @ A ; mlJmin
@ mm sorbent layersG > A 4 mlJmin
; mm sorbent layersG 4 A 53 mlJmin
Be(ore the +a,/le +olution i+ introduced, the +or>ent layer +hould >e
co,/letely )etted )ith +ol?ent and at lea+t a (urther 5 ,in allo)ed (or
equili>ration* *f the sample is introdued without allowing a short period for
e$uilibration% then sharp bands will be formed on the rotor but evaporation in
the solvent olletion hannel will ause slight tailing during fration olletion"
The solvent flow an be redued or turned off during the = min e$uilibration"
The dar! band at the edge of the rotor onsists of sorbent ompletely wetted
with solvent" Other parts of the sorbent are only partially wetted"
55
!bserve the solvent front during the initial solvent introduction" *t should be
irular and onentri for at least the first = m of travel" The front may
beome slightly eentri later due to the large temperature rise that ours as
the solvent meets the sorbent" A front that appears to wobble during the first
few m is an indiation of a worn or bent wi!% an inhomogeneous sorbent%
vapor lea!s around the Teflon lid or an e1essive flow of nitrogen" #ee page
7=% TRO-C<(#HOOT*N)"
.apor lo!s 8bubbles that remain in the pump9 may redue the flow rate when
ambient temperatures are high" Raise the solvent reservoir about @3 m or
hange to less volatile solvents to orret this problem" #ee page 74%
TRO-C<(#HOOT*N)"
After 53A@3 min of operation% ondensed solvent droplets will appear on the
Teflon lid" Warm with the hand to lear slight fogging% tap the Teflon to
oalese larger droplets" Do not increase the nitrogen flow to remove
condensed solvent drops"
/olar solvents do not flow smoothly through Teflon tubing" Remove the output
tube if solvent ba!s up or flows intermittently"
l1T406!CI1: "16 2L!TI1: T32 S"5;L2
+issolve the sample% e"g" 5A@ mg of the test mi1ture 8Appendi1% page ;@9% in a
small volume 83"= A @ ml9 of the eluting solvent" #uitable solvents for the test
mi1ture are heptane A isopropyl aetate% or he1ane A ethyl aetate% both 4G@% or
toluene" *f this is the first run% hoose toluene" The test mi1ture is very soluble
in this solvent and good resolution is easily obtained" Toluene is -. absorbing
and thereby blo!s detetion of -. absorbing samples"
I1T406!CI1: T32 S"5;L2#
5 /ump solvent to the Chromatotron
until the sorbent is ompletely
wetted and allow a further = min
or more for e$uilibration"
5@
@ Ta!e up the sample solution through
the filter and pump input tube"
The sample must be ompletely in
solution" 2ilter or entrifuge
as re$uired"
7 2lush the last traes of the sample
into the filter and tube using
several s$uirts of solvent"
; Return the filter and input tube
to the solvent reservoir"
The volume of solvent used to add the sample is not as ritial as for regular
prep T<C although the onentration should not e1eed about 53K" The high
visosity of more onentrated solutions will distort bands on the rotor" #ample
solution volumes of 5A@ ml 8per mm of layer thi!ness9 are usual but volumes
as large as ;A4 ml 8i"e" up to about 73 ml for a ; mm layer9 will give reasonably
narrow bands" *f a band is broad it will sharpen as the development proeeds"
There is no reason to have initial bands of less than about 7 mm width sine
resolution will not be improved"
*t is possible to introdue the sample after wetting only a small part of the rotor
with solvent" -nder these non e$uilibrium onditions relative Rf values and the
separations obtained may be abnormal" (vaporation in the solvent olletion
hannel will ause slight but usually aeptable tailing of frations"
Introducing Le++ Solu>le Sa,/le+ 8l and @ mm layers only9" *f samples will
not dissolve in a reasonable volume of solvent% wet the rotor ompletely with a
more polar solvent% introdue the sample in this solvent% allow a few minutes
for solvent to drain from the rotor and then dry out the sorbent with an
inreased nitrogen flow 85A5"= <Jmin9" (lution an then be performed with the
desired solvent" -se suffiient of the polar solvent to form a broad bandI a
narrow band may rystalli'e on drying out" The solvent evaporation is very
slow sine the rotor beomes oldI ;=AB= min will be needed" +o not ta!e off
the Teflon lid to speed up evaporationI water will ondense on the old
sorbent"
57
#amples of low solubility an also be handled by the standard method used
with olumn hromatography% that is
introdution as a solution in a small volume of
a polar solvent after wetting the sorbent with
the less polar elution solvent" #uess
depends on minimi'ing the amount of polar
solvent that ompromises hromatography
until diluted"
5a'i,u, and 5ini,u, &lo) 4ate+* )ood resolution will be obtained on
silia gel 8l mm9 with flow rates of @A= mlJmin"
At a flow rate of about =AB mlJmin% depending
on the type of silia% the solvent will flow over
the surfae of the sorbent at the inner edge
ausing strea!ing and loss of resolution for
bands in this area" With lighting from diretly
above% surfae flow is visible as a shiny band
8pulsating if a pump is used for solvent supply9" +ihloromethane and other
high density% low visosity solvents an be used at higher flow rates without
surfae flow"
The ma1imum useful rate of solvent flow varies with the density and visosity
of the solvent but the above rates are appro1imately orret for the low
visosity solvents ommonly used for hromatography" With more visous
solvents% use the test mi1ture 8Appendi1% page ;59 first" A sharp band even
with no resolution% indiates proper solvent flow"
At high flow rates a dar! band appears at the inner edge of the sorbent where
solvent ompletely fills the pores"
There is no minimum solvent flow rate% however% low rates do not improve
resolution and will e1aggerate defets suh as the sloping of bands through @
and ; mm layers"
4a/id Chro,atogra/hy" 2or rapid hromatography inrease the flow rate
after the bands have moved away from the entral area where surfae flow
ours" Alternatively% inrease the diameter of the sorbentAfree area at the
enter of the rotor by mounting in the
oating arbor and turning with a slow
speed stirrer motor while sraping with a
spatula" Removal of @ m or more of the
layer will allow muh higher solvent flow
rates without surfae flow" 2or e1ample%
the test mi1ture 8page ;59 separates within
= min using silia gel and toluene at double the normal flow rate"
5;
Interru/ting Sol?ent &lo)* #olvent flow may be shut off for a onsiderable
time during elution with no effet on the resolution ultimately obtained" *f the
flow is to be off for more than @3 min% inrease the nitrogen flow to evaporate
the solvent from the sorbent" The bands of ompounds on the dry rotor will not
spread by diffusion" The new solvent flow will sharpen the bands"
3ea?y Loading* 2or heavy loadings% dissolve the mi1ture in several ml of
solvent" Conentrated solutions 8Ll3K9 produe eentri bands 8due to the
inreased visosity9" #light eentriity is normal for heavy loading"
Light Loading" .ery small $uantities of -. absorbing mi1tures may not be
detetable when spread out as irular bands on a rotor" More onentrated
bands are obtained if the solution of the mi1ture is applied diretly to the dry
sorbent on the rotor as a single spot or a series of spots in a short ar" To
observe the bands in -. light% turn off the solvent flow and stop the rotor"
Choose a solvent whih gives a low Rf so that e$uilibrium onditions will be
attained before the ompounds are eluted" Resolution of mi1tures applied as
single spots or short ars is better than that from the omplete irles of the
normal proedure"
-se thin sorbent layers to maintain higher onentrations of small samples"
#ee the sraper blades in the CHROMATOTRON /ART# <*#T"
62T2CTI01 0& !< "BS04BI1: C05;0!16S 01 T32 40T04
A -. lamp 8e"g" a Mineralight% short wave% -./ *n"9 held over the Teflon lid of
the Chromatotron will reveal bands of -. absorbing ompounds" Cands will
be visible diretly under the lamp and also for some distane around the rotor
due to the delay between absorption of -. and emission of visible light from
the phosphor" +etetion at short 8@=; nm9 wavelengths is the most generally
useful"
A band of apparently even density will have most material near the enter and
very little at the leading and trailing edges" Two ompounds that are ?=K
separated may therefore appear as a single broad band"
2or detetion at the output tube% spot the eluate on a T<C plate held under a
-. lamp" The la! of pressure at the output and the presene of gas bubbles%
prevents diret onnetion to a -. monitor"
#ome hand lamps do not produe suffiient -. light% espeially after long use"
This problem is easily solved by removing the light filter 8whih absorbs most of
the -. light9" #hading the e$uipment from room lights will also give an
improvement" A further enhanement an be obtained by turning off the
solvent flow and stopping the rotor" *f detetion diffiulties are due to a small
sample si'e% see <ight <oading% above"
5=
#olvents must not absorb -. light" #ome suitable -. transparent solvents are
he1ane% heptane% dihloromethane% ether% tetrahydrofuran% aetonitrile%
methanol% ethanol and isopropanol" (thyl aetate absorbs -. light but is
usable in most ases" -se dihloromethane if the hoie is between
dihloromethane and hloroform" Cen'ene is a ommon impurity in some
grades of he1ane and ethanol and will interfere with detetion" The ease of
detetion of a sample in a partiular solvent an be he!ed by ordinary T<C"
Observe the stillAwet T<C plate in -. light"
I( acetone A!< a>+or>ingB i+ u+ed a+ a clean-u/ +ol?ent, it ,u+t >e
re,o?ed co,/letely (ro, the /u,/, tu>ing and +or>ent* /referably% airA
dry the rotor" A solvent wash an be used to remove aetone but surprisingly
large volumes are re$uired to remove the last traes from the sorbent" A -.
absorbing test substane with a high Rf% suh as phenanthrene or other
aromati hydroarbon% provides a onvenient means of he!ing that all is well
before introduing more valuable ompounds"
62T2CTI01 0& C0L04L2SS !< T4"1S;"421T C05;0!16S
Compounds whih annot be deteted by -. absorption should be analy'ed
by onventional T<C after olletion" )radient elution overing a wide polarity
range will ensure that all ompounds have been eluted"
Compounds may be deteted at the output tube by spotting on a lean ground
glass stopper" (vaporation of the solvent leaves an observable residue"
#ome ompounds la!ing hromophores an be deteted on sorbents with
long wavelength 87>= nm9 phosphors 8H"," +esai% ("R" Trumbull and #"W"
/elletier% M" Chromatography 5?4> 3 ;7?9" Cands on a rotor an be
visuali'ed% inonveniently% by evaporating the solvent and applying iodine 8H","
+esai% C"#" Moshi and #"W" /elletier% M" Chromatography 5?4= 322 @@79" Most
of the methods used for detetion of -. transparent ompounds on regular
T<C plates should be appliable in the Chromatotron" These inlude -. light
with berberine 8<" Mamlo!% M" Chromatographi #iene 5?45 19 =79 in the
sorbent and 8for polar ompounds9 he1ane 8," #uyama and #" Adahi% M"
Chromatographi #iene 5?4B 25 5739"
Chromophores an be designed into the intermediates of a reation se$uene
by inluding proteting groups with an aromati system e"g" ben'oi esters or
trityl ethers"
&4"CTI01 C0LL2CTI01
The narrow bands produed by the Chromatotron re$uire that frations be
small" Colletion in disposable test tubes or ulture tubes allows a deision on
how to ut or mi1 frations to be made later after analytial T<C" Colletion in
a small number of (rlenmeyer or roundAbottom flas!s inevitably remi1es parts
of lose frations"
5>
The output tube should be within the test tube to prevent loss by splashing as
the bubbles in the eluate burst"
A small amount of a olored ompound with a high Rf% e"g" a'oben'ene or the
test mi1ture 8page ;@9% added with the mi1ture to be separated% will mar! the
Fsolvent frontFI olletion of frations an then be delayed until this has been
eluted" 7hen collecting !< a>+or>ing co,/ound+, continue to collect
+e?eral (raction+ a(ter the !< a>+or>ing >and ha+ a//arently /a++ed
(ro, the rotor* The last part of the band is not easily seen and signifiant
amounts of ompounds an be missed" The trailing edge of a band an be
deteted by spotting the eluate on a T<C plate and observing the residue in -.
light after the solvent has evaporated"
#ine the separations are ompleted in a very short time% automati fration
olletion has few advantages over hand olletion" *f a fration olletor is
used% hoose drop ounting or time mode" #mall frations will partially remi1 in
olletors using a siphon tube" The output from the Chromatotron will not pass
through long lengths of narrow tubing" -se tubing with a bore of about
5J5>F 85"= mm9 to onnet to a fration olletor and !eep the gradient to a
minimum" The base of the Chromatotron an be removed to allow positioning
over a fration olletor"
CL2"1-!; "16 42:2124"TI01 0& T32 S04B21T L"@24
After eah separation or when sorbent layers have beome ontaminated with
polar ompounds% a leanAup with a polar solvent suh as aetone is re$uired"
-se at least ;3 ml per mm of layer thi!ness" At the start of the leanAup%
unsrew the inlet about a $uarter turn to move the wi! and solvent flow loser
to the enter of the rotor where traes of samples may remain"
The leanAup solvent an be removed from the sorbent by drying in the open
air% allowing suffiient time 8at least l@ hr for l mm% @; hr for @ mm and ;4 hr for
; mm layers9 for evaporation of the solvent and of water that ondenses on the
old sorbent" A metal Nheat sin!O under the rotor will enourage the
evaporation" +rying with slight heating 8the lamp and bu!et on page @B is
effetive9 may also be used 8and will be essential in humid limates9 after the
solvent has co,/letely evaporated" *n ase some solvent is still present% do
not u+e an o?en* #ake care to remove all traces of acetone from the solvent
reservoir and tubing if $% absorption is to be used later for detection.
5B

CleanAup solvent an also be removed by a solvent of intermediate polarity
suh as dihloromethane 8at least ;3 ml per mm layer thi!ness9 followed by
an e$ual volume of the solvent to be used in the separation" The diret
transition from aetone to a solvent of low polarity suh as he1ane A ethyl
aetate 8?Gl9 re$uires muh larger volumes of solvent 8at least l@= ml9" High
Rfs% broad bands and poor resolution will result from the use of insuffiient new
solvent for removal of the leanAup solvent"
2or a more rapid removal of leanAup solvent from l or @ mm layers% first
evaporate with nitrogen 8l A l"= <Jmin9 then follow with the new solvent" An
overnight nitrogen flow of about @=3 mlJmin will also remove solvent"
Methanol may be used to remove very polar impurities from sorbent layers"
Removal of the methanol by other solvents is a slow proess% preferably dry
the rotor in the open air" 2or most separations a silia gel layer leaned one
with methanol will give sharper bands% however% repeated use of l33K
methanol will slowly remove the gypsum binder and hange the nature of the
sorbent" Relative Rf values of the omponents of a mi1ture will also hange"
When hromatography and leanAup have been ompleted and no further
separations are ontemplated% wash out hloroform% dihloromethane% aetone
and aetonitrile with he1ane or ethyl aetate or leave a slow stream of nitrogen
passing into the Chromatotron to redue the possibility of solvent damage to
plasti parts"
5!LTI;L2 62<2L0;521T
#harpen broad or irregular bands on l and @ mm layers by multiple
development" Turn off the solvent flow and wait a few minutes for the flow at
the output to derease to a low rate" *nrease the nitrogen flow to 5A l"= <Jmin
for ;=A>3 minutes or more to evaporate the remaining solvent from the
sorbent" The evaporation is a slow proess sine the rotor beomes very old"
+erease the nitrogen flow and restart the solvent addition" Cands will beome
onsiderably sharper" Multiple development is less effetive when Rf values
are low" Do not remove the #eflon lid to evaporate solvent& water will
condense on the cold sorbent.
42C@CL2
Reyle is a simple strategy for inreasing the resolving power of the
Chromatotron" 2or a trial run use the test mi1ture 8Appendi1% page ;59 with
he1ane A ethyl aetate 8>G;9" This solvent mi1ture% ontaining a large
proportion of the polar omponent% gives inomplete separation in one pass
and omplete separation on reyle"
54
2or reyle% proeed as in normal elution " until the bands to be reyled are
about 5 m from the edge of the sorbent then remove the solvent input tube
from the solvent reservoir% B" After waiting about l3 se to redue the amount
of solvent in the pump and Chromatotron% onnet the filter 8with otton9 to the
output tube 8with detahable part removed9%C" The pump will reyle solvent
from the Chromatotron for about l min then both solvent and vapor will reyle"
" B C
*f the l3 se delay at stage B is omitted% solvent may ba! up slightly in the
olletion hannel ausing some remi1ing of bands" #he solvent flow rate must
be held constant for some time before recycle commences to prevent surges
of solvent flow.
When the bands of interest have been returned to the rotor it is preferable to
return to mode " sine in mode C evaporation will slowly redue the amount of
solvent available for reyle"
Cands will be disouragingly diffuse when newly reyled ba! to the sorbent"
However% on further development they will sharpen up onsiderably" After one%
two or three stages of reyle% depending on the loading and the tailing
tendeny of the partiular ompounds% the bands will beome so broad that
further reyle will ause the head of one to ath up with and overlap the tail
of another" At this stage the band or bands should be eluted or if reyle is to
ontinue they must be sharpened by multiple development 8page 549" *f the Rf
of the bands is 3"= or more% multiple development is very effetive for band
sharpening% allowing an unlimited number of reyles to be performed"
C0112CTI1: C3405"T0T401S I1 S24I2S
Resolving power and apaity an be inreased by onneting two 8or more9
Chromatotrons in series" /ump the output of one to the input of the ne1t"
Ca!Aup of solvent in the first Chromatotron an be prevented by setting the
intermediate pump at a higher flow rate so that both solvent and some vapor
are pumped"
5?
CCCCCCCCCCCCC0"TI1: 40T04S 7IT3 S04B21TS
I1T406!CTI01
#orbent layers on rotors are produed by asting sorbentAbinder mi1tures
followed by sraping down to 5 mm% @ mm or ; mm thi!ness with a rotating
sraping tool" More binder is re$uired than in onventional analytial T<C"
Calium sulfate hemihydrate 8plaster of /aris% dried gypsum9 is the most
fre$uently used binder" #etting is very sensitive to the pH% the temperature%
and to the presene of other solids or solutes"
#atisfatory binders for C54Asilia and other hydrophobi sorbents are not
available"
Rotors are made of regular glass% not a heat resistant type" +o not submit
them to unneessary thermal sho!s" Rotors with wet layers are li!ely to ra!
at the enter if plaed in an oven at temperatures above =3PC"
Reipe 5 8page @59 forms silia gel layers that satisfy ?=K of the needs of
most hemists" Other reipes are useful in speial ases"
S04B21TS, BI1624S "16 ;30S;304S
#igmaAAldrih item no"
#ilia gel% T<C standard grade 8no binder9 with fluoresent
indiator
@44=4>
2luoresene indiator green @=; nm 8'in siliate9 3@==;
/olyethylene glyol 4333 @3@;=@
Aluminum o1ide #A* #ientifi Adsorbents *n" Cat" no" 3;7;;A5
Aluminum o1ide )2A@=; ("Mer!% *tem 53?@
#igmaAAldrihG sigmaAaldrih"om
#ientifi Adsorbents *nG saiadsorbents"om
Calium sulfate hemihydrate 8plaster of /aris% dried gypsum9 is available from
various hemial suppliers" /laster of /aris from hardware stores is usually of
better $uality"
(" Mer! produts are available through .WR #ientifi and other laboratory
suppliers"
@3
42CI;2S
Chec= the quality o( the calciu, +ul(ate he,ihydrate >inder# #tir a =g
sample and = ml of water with a spatula in a @= ml bea!er then mi1
ontinuously by swirling" The mi1 should thi!en after about BA5; min to the
point where it will not move when tilted" Thi!ening outside of this time interval
indiates the presene of alium sulfate dihydrate impurity% that will ma!e
rotor oating diffiult" *f sorbent layers ra!% try another soure of plaster of
/aris or regenerate at about 5>3 for 4 hr"
Cooling of the water in the reipe redues air pressure buildAup during the
sha!ing of the ontainer" A thi!% reamy but pourable mi1ture is re$uired"
Amounts of sorbents and binders are given in grams% amounts of water in ml
and temperatures in C"
Sugge+ted 5i'ing 9ar SiDe+
<ayer thi!ness 1 ,, 2 ,, 4 ,, $ ,,
Mi1ing 0ar si'e @=3A;33 ml 733A;33 ml ;33A>33 ml B=3A5533 ml
2or easy mi1ing% the 0ar should be not more than B3K full with the dry sorbent
and binder"
4eci/e 1, Silica :el TLC Standard :rade - :y/+u,
#ilia gel T<C standard grade 8ontains @=; nm fluoresent indiator9 is the
most useful sorbent"
1 ,, 2 ,, 4 ,,
#ilia gel% T<C standard grade 77 ;= B=
Calium sulfate hemihydrate 57 54 73
Water at 3A53 4@ 55@ 54B
Allow the layers to set for at least 5 hr 8overnight O,9 before drying"
#ee page @; for the mi1ing and pouring proedure"
@5
4eci/e 1B, Silica :el TLC Standard :rade - :y/+u,, $ ,, Layer+
$ ,,
#ilia gel% T<C standard grade 57B
Calium sulfate hemihydrate ==
Water at 3A53 7;7
Allow the layers to set for at least 5 hr 8overnight O,9 before drying"
#ee page @; for the mi1ing and pouring proedure"
The 4 mm layers are not reommended" +rying of the layers re$uires several
days" The drying out of solvents from the layers after use is also a long
proess" A ; mm layer used twie will give the same results with less trouble"
When oating with the 4 mm reipe% use 5O 8@"= m9 mas!ing tape around the
rotor and also around the entral metal dis! of the oating arbor" A long%
strong spatula is re$uired for the initial stirring"
#olvent flow rates for 4 mm layers should be the same as for ; mm layers"
Higher flow rates may ause splashing"
4eci/e 2, "cidi(ied Silica :el-:y/+u,
Aidifiation of silia gel layers will prevent tailing of aidi samples" Only very
wea! aids suh as ammonium sulfate 8giving a pH about ;"=9 an be added
to the reipe" O1ali and itri aids inhibit setting of the binder"
-se reipe 5 with ammonium sulfate 85 mm A 3";=g%
@ mm A 3">=g% ; mm A 5"@g9 dissolved in the water"
Allow ;hr for setting"
Ammonium sulfate is not eluted by aetone but is removed by methanol"
*f a stronger aid is re$uired% prepare layers from reipes 5 or > and add aeti
aid to the eluting solvent"
@@
4eci/e 3, Silica :el - Sil?er 1itrate - :y/+u,
2or the separation of olefins% f" <"M" Morris% Chem" and *nd" 85?>@9 5@74"
-se reipe 5 with silver nitrate 85 mm A @g% @ mm A 7g%
; mm A =g9 dissolved in distilled water"
Allow ;hr for setting" The finished layers are rather soft and will slowly dar!en
if stored in light"
He1ane A ethyl aetate should be used for elution and ethyl aetate for leanA
up" #ilver nitrate is eluted by solvents ontaining methanol% tetrahydrofuran or
aetone" -. detetion is not effetive in the presene of silver nitrate"
4eci/e 4, Silica :el - ;olyethylene :lycol - :y/+u,
2or partition hromatography of non polar samples% f" M"H" +hont et al"% M"
Chromatography% 85?B59 0 @>=" #ee page 75 for a disussion"
-se reipe 5 or @ with polyethylene glyol 4333 or >333
85 mm A >"=g% @ mm A ?g% ; mm A 5=g9 dissolved in the water"
Allow ; hr for setting" <ayers are rather soft"
2or elution use only he1ane% ether% and aetoneI polyethylene glyol is soluble
in most other solvents" Only non polar hydroarbon solvents give good
resolution"
4eci/e 5, "lu,inu, 0'ide - :y/+u,
Type #A* aluminum o1ide is preferred" Aluminum o1ide )2 must be
regenerated 8heat at 5>3 for 7 hr9 to avoid unpreditable results"
1 ,, 2 ,, 4 ,, 1 ,, 2 ,,
Aluminum o1ide Type #A* == ?3 5>3 Type )2 == ?3
Calium sulfate hemihydrate 57 @@ 74 53 5>
2luoresent indiator 3*= 5 5*= A A
Water at 3A53 == ?3 5>3 >@ 533
#ee page @; for the mi1ing and pouring proedure" The mi1ture% partiularly
for 5mm layers% does not flow freely over the rotor" #pin the oating arbor and
rotor by hand to enourage flow to the edge" *f neessary% pi! up and tilt the
rotor" 2inally bump the rotor and arbor" #etting re$uires at least ; hr"
@7
4eci/e , Silica :el - :lue
Glue-bound layers are stable to very olar !"n#lud"n$ a%ueous& solvents'
1 ,,
#ilia gel T<C standard grade 73
)lue @*=
Water B4
#ee page @? for the oating proedure and a disussion"
4eci/e+ (or &lori+il and Cellulo+e
2lorisil layers an be made using glue as in reipe >" The layers are very
al!aline and bands NtailO"
Cellulose layers are diffiult to prepare and do not perform well"
C0"TI1: 40T04S !SI1: 42CI;2S 1-5, :@;S!5-B0!16 L"@24S
Introduction
The beginner should first prepare a 5 mm layer of silia gel from reipe 5
8page @59 for whih the proedure is least ritial" Typially% the proessing
time for the first rotor oated is e1essive and a less than perfet layer is
produed" However% the tehni$ue is easily mastered and the seond rotor
prepared is usually a suess"
Cefore starting the oating proedure obtain a lear a!e over% ardboard bo1
or plasti bowl to over the rotor while the layer is setting" Have a strong
spatula ready for stirring"
@;
"ll rotor+, ne) or u+ed, ,u+t >e thoroughly cleaned )ithin 1E2 hr o(
coating* 6o not rely on gla++ )a+hing +er?ice+* &ollo) the cleaning
in+truction+ care(ullyF 6etach,ent o( +or>ent layer+ )ill >e a +eriou+
nui+ance i( rotor+ are not +cru/ulou+ly clean*
Cleaning and Setting !/ the 4otor &or Coating
Mount the rotor ground side up in the oating arbor and tighten gently by
turning the edge of the rotor while holding the plasti handle" The rate of
aidental rotor brea!age will be high if the arbor and handle are not used
during leaning"
Thoroughly +cru> the u//er +ur(ace )ith
)ater and "Ga' or +i,ilar a>ra+i?e
hou+ehold clean+er /o)der, u+ing a )ad
o( /a/er to)el+, nylon +couring /ad or a
+,all +ti(( >ru+h" Wash well and he! for
effetive leaning by allowing the water to
drain off for about 5= se" *f separate drops
of water form on the rotor then the leaning is
not omplete and should be repeated"
6ry )ith /a/er to)el+ only" -se at least
two% wiping first the leaned upper surfae then the other parts of the rotor and
arbor" A final wipe of the dry rotor with a dry towel will remove lint" +o not dry
with aetone or by heating in an oven"
Attah 7J;F 8@ m9 mas!ing tape
8preferred9 or other adhesive tape
8ellulose tape may be used but will
leave adhesive on the glass9 to the
edge of the rotor as shown" /ress
the tape on firmly to the edge and
underside of the rotor" Attah e1tra
piees of tape as re$uired" 2or easy
removal of the tape later% form a tab by bending over the tape end"
/lae on a rigid le?el surfae overed by a piee of thin felt% loth or a single
layer of a paper towel% taped down" The surfae used must be free from
vibration aused by nearby e$uipment" -se grommets or a short length of
rubber tubing on the rod of the arbor as a finger grip"
5i'ing and ;ouring the 4eci/e
Weigh out the sorbent and binder into a preserve or pi!le 0ar or other wideA
mouth ontainer with a srewAon ap giving a li$uidAtight seal" (rlenmeyer
8onial9 flas!s are inonvenient" 2or easy mi1ing the 0ar should be not more
than B3K full of the dry sorbent and binder" -sual 0ar si'eG 5 mm layers%
@=3A;33 ml% @ mm% 733A;33 ml% ; mm% ;33A>33 ml"
@=
Measure out the water% ool% in a refrigerator and
add to the 0ar" The following stages must be
ompleted within 7 min sine the mi1ture will
begin to set within 7A53 min"
#tir the mi1ture with a spatula until most dry
po!ets at the bottom and sides of the 0ar have
been wetted 8about 53 se9" Remove the spatula 8ignore the mi1ture that is
attahed9 then over and sha!e very vigorously end to end for 53A@3 se"
Turn the rotor slowly 85J@ A 5 revolutionJse9 by the entral
rod and pour the mi1ture in a ontinuous stream in
overlapping irles lose to the entral metal dis!" ,eep
the 0ar lose to the rotor to minimi'e the formation of
separate drops of the mi1ture" #light agitation will
enourage thi! mi1tures to flow" Touh the mouth of the
0ar to the metal dis! of the arbor to remove the last
hanging drop"
*f suffiient water has been used% ?=K of the mi1ture will flow
readily out of the 0ar"
)rip the enter rod of the arbor and raise the rotor about
5 m then lower% bumping the handle below the rotor
against the felt 8or loth9 overed surfae" +o not turn
during the bumping" Repeat about 7 times" Cumping
li$uefies the mi1ture releasing air bubbles% allowing
gravity to spread the mi1ture and smooth out
inhomogeneous parts"
Co?er the rotor )ith a clear ca=e co?er or
card>oard >o' A/rotection (ro, dra(t+B
and allo) the layer to +et (or at lea+t 1 hr
Areci/e 1B or 4 hr Areci/e+ 2-5B* Overnight
setting is onvenient"
Remove the mas!ing tape after the setting is omplete" The rotor an remain
in the oating arbor for air drying of the layer"
6rying the Sor>ent Layer
The traditional FativationF of regular T<C plates by heating at 533P is only a
reversible drying" The sorbent will $ui!ly reAe$uilibrate with atmospheri
moisture unless the plates are stored in a dry atmosphere" 2or Chromatotron
rotors% air drying alone will give satisfatory FativityF in dry limates" *n humid
limates oven drying at ;3A=3P will be re$uired"
@>
"ir 6rying" It is a common mistake to assume that a layer is dry when the
surface appears to be dry. 'ir dry to constant weight (.)g *weigh the
complete rotor+arbor assembly,. Rotors oated with reipe 5 or 5C an be
dried in the open with no heating and no speial preautions"
#light heating with a lamp as shown will provide a more rapid drying% espeially
useful for the thi!er sorbent layers"
With a lamp heater% onstant weight is usually reahed in about 5@ hr for a
5mm layer% within about 7> hr for a ; mm layer
#ome types of silia gel 8e"g" Mer! silia gel /2% at" no" BB;?9% that were
previously suggested for use with the Chromatotron% are sensitive to drafts"
NonAirular bands were formed on layers dried in a unidiretional air flow"
0?en 6rying* #rape off a irle of sorbent around the metal dis! of the arbor
with a spatula or piee of wire% to allow removal of the rotor from the arbor"
-nsrew by turning the edge of the rotor while holding the handle of the arbor"
#he glass will crack if the rotor is not removed from the arbor before heating.
Oven dry at ;3A=3" -otors may crack if a wet layer is placed in an oven at
higher temperatures" 'lso, layers become fragile at temperatures above .(.
Any oven that is not sealed will be suitable" Dry to constant weight (.)g,
about > hr for 5 mm layers% 53 hr for @ mm and @3 hr for ; mm" The progress
of the drying an be he!ed by touhing the underside of the rotor with the
fingers while in the oven" The glass will remain ool until most of the water has
evaporated"
Although air drying of sorbent layers is most onvenient% oven drying gives
better adhesion to the glass rotor and is preferable if large volumes of
methanol are to be used for elution"
4otor+ that ha?e >een u+ed )ith +ol?ent+ Ae+/ecially /olar +ol?ent+
+uch a+ ,ethanolB +hould not >e /laced in an o?en* !+e the la,/
heater a>o?e, a(ter all +ol?ent ha+ e?a/orated*
@B
SC4";I1: S04B21T L"@24S
Allow oven dried rotors to ool ompletely before sraping" Hot rotors may be
slightly distorted" Replae the rotor in the oating arbor% removing if neessary
any loose sorbent between the rotor and the arbor" Hold the rotor vertial and
plae the sraping tool 8with l% @ or ; mm blade9 on the shaft as shown" Turn
the sraper lo!wise" To avoid hipping the sorbent% apply only slight
pressure" This is a noisy operation H
Continue sraping until stage A is reahed with @ hannels sraped ompletely
down to the glass" *f the sraping tool an be turned only with diffiulty% hold
the outer end instead of the handle" *f the sraper bounes over the sorbent%
forming radial ridges% again hold the end of the tool" Alternatively% srape in
reverse for a few turns"
Change to the finishing sraper to remove unwanted sorbent at the edge and
enter 8stage A A C,"
@4
Remove the rotor from the oating arbor and use the end of a sraper blade to
hisel off any sorbent remaining at the enter" Rub off sorbent at the edge or
on the underside of the rotor"
Clow off loose sorbent dust from the surfae into a fume hood" Clowing by
mouth or with ompressed airJnitrogen from a pinhed rubber tube is
satisfatory" Most eletri handAheld blowers are too wea!" Any dust
remaining on the rotor will appear on the Teflon lid or in the solvent olletion
hannel"
The finished sorbent layer may have a few irular ruts and several bubble
pits" These do not affet performane" #uff mar!s and the loss of small
piees of the sorbent at the edge% have a negligible effet" <oose piees of
sorbent should be removed sine they may detah later"
#raper blades are easily sanded down by rubbing on a sheet of emery paper"
A ; mm blade% for e1ample% an be ad0usted to 7"? mm to allow sraping of a
layer that is not $uite the intended ; mm thi!ness" Ad0usted blades an also
be used to lean off a thin layer of sorbent from an old or damaged layer"
ST04":2 0& C0"T26 40T04S
Rotors stored in the open will pi! up volatiles from the lab air but most of
these wash out in the first few ml of solvent" Cefore mounting rotors in the
Chromatotron% blow off dust that will otherwise gather around the outlet hole in
the olletion hannel"
Old rotors develop slight pH differenes between the upper and lower layers of
sorbent" Only the separation of aidi or basi samples is affeted"
In (o"st #l"(ates) store rotors "n a #ab"net *"t+ dry"n$ a$ents or a slo* strea( o, dry
a"r-n"tro$en or re-dry *"t+ t+e la( or la(-bu#.et asse(bly be,ore use'
<abel rotors at the edge with a penilG
C0"TI1: 40T04S !SI1: 42CI;2 , :L!2-B0!16 L"@24S
A variety of ommon a$ueous late1 glues are satisfatory adhesives for silia
gel 8reipe >% page @;9" The most useful are clear Nsilioni'edO 8or Nwith
silioneO9 aryli late1 sealants for !ithen and bathtub aul!ing sold in
s$uee'e tubes by hardware stores"
@?
Weigh out the sorbent into a @=3A733 ml 0ar or flas!% add all but 53 ml of the
water and stir with a spatula until ompletely wetted" #uspend the glue in 53
ml of water% by magneti stirring or with a spatula% add to the wet sorbent and
stir" The mi1ture may thi!en initially but will thin out within a few minutes"
Cover with aluminum foil 8without removing the spatula9 and allow the mi1ture
to stand for at least 5J@ hr" *f many bubbles are trapped in the mi1% stir gently
and bump the 0ar to allow bubbles to rise and brea!" *f the mi1 is too thi! for
easy stirring% add water in @ ml inrements"
A tape edge annot be used around the rotor when pouring glueAbound layers"
The tape detahes during the many hours that the mi1ture remains fluid" With
e1periene and some lu! sorbent layers an be poured without the tape edge"
A more onsistent solution is shown in the diagram on the ne1t page"
)ive the mi1ture a final stir and pour in the usual way" Cump the rotor and
arbor until the mi1ture flows to the edge at all points" *f the mi1ture is too thi!
to flow% pi! up the rotor and tilt"
Allow the layer to dry to onstant weight in the open 8do not over with a bo19"
The drying time is about ;4 hr in dry limates% The lamp heater an be used
after about 5@ hr when the layer has partially set After air drying% remove the
rotor from the arbor and plae in an oven and heat at =3 for about 5 hr"
Wash the sraped rotor in the Chromatotron with at least 5@3 ml of aetone to
remove large amounts of unpolymeri'ed glue"
1or,al ;ha+e Chro,atogra/hy" #ilia A glue layers perform well in normal
phase operation% e"g" the test mi1ture 8page ;59 in toluene" Cands of
ompounds are usually narrow and onentri" The layers do not detah from
the glass even with a$ueous solvents"
4e?er+ed ;ha+e Chro,atogra/hy" A very limited form of reversed phase
hromatography is possible on silia A glue layers" The test mi1ture will
separate in reverse order with aetone A water 85G59" Apply the test mi1ture as
a filtered saturated solution 8about @ ml9 in aetone A water 8>G;9"
A$ueous solvents s!ate over the surfae when applied to a dry sorbent" #tart
with 533K organi solvent then hange to the a$ueous solvent" As the
perentage of water in the solvent is inreased% a point is reahed where
solvent begins to splash around and the utility of the Chromatotron is lost"
Only non polar samples separate well with this sorbent under R/ onditions
and resolution does not approah that obtained by regular T<C on silani'ed
layers"
73
4otor Set-!/ (or Slo) Setting Layer+
Mas!ing tape in ontat with a wet layer for more than about @3 min will detah
from the rotor and allow the mi1ture to lea! out" The rotor setAup below
prevents lea!age"
5 Clean and dry a
rotor" Remove from
the oating arbor and
dry the entral area"
@ /lae another rotor on the
oating arbor handle% over
with a s$uare sheet of
aluminum foil" /ress the
entral area with the fingers
to mar! the enter hole then
piere with the oating
arbor"
7 /lae the lean rotor on
top% insert the oating arbor
and srew in" 2ull tightening
re$uires touhing both rotors
8outer = mm onlyH9 to allow
turning the rotors while
holding the handle" Cut the
aluminum foil to a rough
irle e1tending about @ m
beyond the rotor edge"
; /ush the aluminum foil up
and apply mas!ing tape in
short 853 m9 piees" The
tape is for stiffening only% not
for sealing"
= /ress the foil and tape
together forming a neater
edge"
;"4TITI01 C3405"T0:4";3@, 42CI;2 4
/artition of ompounds between a moving solvent and a li$uid stationary
phase retained by silia gel provides a separation method $uite different from
adsorption hromatography" /artition is partiularly suessful for the
separation of mi1tures of ompounds that differ in the ratio of polar to non
polar groups% suh as homologs% and for separating unrelated ompounds
whih by hane have the same Rf by adsorption hromatography" Only
hydroarbon solvents give good resolution
75
Reipe ;% page @7% for partition layers ontaining polyethylene glyol is based
on the data of M"H" +hont% M"C" +e Ceauveser and )")" ,ui0pers% M"
Chromatography l?Bl 0 @>="
/olar solvents are also suitable as stationary phases" 2or e1ample%
hromatography by partition between aetonitrile and he1ane may be
performed as follows" Run in aetonitrile% previously e$uilibrated with he1ane%
onto a 5 mm silia gel layer in the Chromatotron until the solvent front is about
halfway aross" +isonnet the solvent delivery system and remove all
aetonitrile from the tubing% inlet and pump" After allowing the aetonitrile
stationary phase to e$uilibrate on the rotor for at least 73 min% run in he1ane%
previously e$uilibrated with aetonitrile% and apply the sample% also in he1ane"
The two bands from the test mi1ture 8page ;59 will be sharper and more
ompletely resolved by partition than by adsorption hromatography"
C0442CTI1: B"16 SL0;2
When @ and ; mm layers are prepared from reipes with ertain silia gels
8e"g" Mer! silia gel /2% at" no" BB;?9 or with an e1ess of water then bands
of ompounds will slope as shown"
Cands will broaden and separated bands may
remerge" /rodut will appear at the output tube
before the observed band on the surfae has
reahed the edge of the rotor" #he slope is
more pronounced at low flow rates and with
high density solvents.
To redue band slopeG
Avoid hloroform 8high density solvent9"
*nrease flow rates"
Redue the amount of water in the reipe"
Cool the water and sorbent"
7@
CCCCCCCCCCCCCCCCCC5"I1T21"1C2 "16 42;"I4S
2L2CT4IC"L C0112CTI01S
(<*NCO motor 55=. Model (<*NCO motor @@3. Model
S0L<21T ;!5;S
#ee page 74% TRO-C<(#HOOT*N)% and the literature provided with the
pump" The following routine maintenane is suggested for model
RH#YOO#TY<2 pumps at one year intervals or whenever the pump head or
motor beome stiff"
Remove the pulley and apply an automotive type oil to the one visible motor
bearing" Wipe off e1ess oil and replae the pulley and drive belt"
77
"69!STI1: T32 5"I1 <2SS2L
*f the rotor support ollar touhes the main vessel% loosen the four srews
holding the vessel to the metal frame of the Chromatotron% ad0ust the vessel
and retighten the srews" The rotor support ollar an also be raised slightly"
T32 T2&L01 LI6 - 5"I1 <2SS2L S2"L
The Teflon lid must seal well to the main vessel" *f the lid is held up by
damage to the reess in the main vessel% use fine sandpaper to sand it down"
42;L"CI1: T32 40T04 S!;;04T C0LL"4
*f the rotor support ollar 8diagram page @9 is removed% note that on
replaement% the two set srews must be aligned with the flats on the motor
shaft" *f the rotor support ollar and the shaft ollar are not fully lo!ed in
position% they will move when the endplate is srewed in% ausing the shaft to
press against and brea! the glass rotors near the enter hole"
T32 &2LT S2"L
The rotor support ollar passes through a felt seal 8diagram page @9 below the
main vessel" +eposits of organi materials on the felt may ause it to adhere
to the rotor support ollar% produing a rubbing sound" No maintenane is
re$uired unless the adhesion auses a slow start up of the motor"
The deposits an be removed by holding the Chromatotron with the plane of
the main vessel vertial while applying solvent to the upper edge of the felt%
allowing it to pass through and run off the lower edge" -se a range of solvents
but finish with a hydroarbon" ,eep solvents away from the motor bearings"
-nplug the Chromatotron before starting this operation"
72"4 "16 T2"4
The original prototype Chromatotron was retired% still in wor!ing order% after 53
years of daily use" The Chromatotron motor is rated for ontinuous duty and is
e1peted to have a long life" /umps are more sub0et to wear and damage
from abuse"
7;
CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT40!BL2S300TI1:
;40BL25 C"!S2 C!42
*rregular or eentri solvent
front and bands" Most evident
with -. illumination"
5 sorbent mi1ture prepared and poured
too slowly% partly set during pouring"
@ #orbent layer not proteted
from drafts during setting"
Cover with ardboard bo1 if the
sorbent has gypsum binder"
7 -neven drying of Mer! silia gel /2" -se the lampJbu!et dryer page
@B or turn rotors during drying"
-se silia gel standard grade"
; #orbent layer not ompletely dried" +ry to onstant weight" *n
humid limates use an oven or
a lamp 8page @B9"
= *nlet wi! is too short% bent up or of
inorret material ausing solvent to
flow down wi! as separate drops"
Change or ad0ust wi! 8pages
=A>9"
> #urfae used for pouring and setting
of sorbent suffers from vibration"
Test with bea!er of water"
.ibration forms visible waves"
B Home made glass rotors are not flat"
4 #olvent mi1ture has omponents of
e1treme polarity range% e"g" he1aneA
aetone" #olvent separates into two
phases visible as dar! spots at inner
edge of sorbent"
-se less e1treme range% e"g"
he1aneAethyl aetate"
? .apor lea!s due to dust at the
edge of the Teflon part 8either side9
of the lid"
Clean lid and reess with a
moist paper towel"
53 .apor lea!ing out of disonneted
nitrogen inlet"
Croad bands"
#ee also radial strea!ing
8below9"
5 Overloaded"
@ CleanAup solvent not removed
ompletely"
7 #ample solution has undissolved
material in suspension"
2ilter or entrifuge sample
solution"
; Cands slope through @ or ; mm
layers% espeially with dense solvents"
#eparated bands may remerge"
Common problem with Mer!
silia gel /2" #ee Correting
Cand #lope% page 7@" -se
#tandard grade silia gel
= Heat from nearby steambaths%
hotplates% et" disturbing e$uilibrium
onditions"
Move the Chromatotron to a
hood" Cool with a small fan"
-se less volatile solvents"
7=
;40BL25 C"!S2 C!42
Cands separate then merge or
broaden% espeially with dense
solvents on @ or ; mm layers"
Cands slope through the layer" Common problem with Mer!
silia gel /2" #ee Correting
Cand #lope 8page 7@9" -se
standard grade silia gel"
Croad initial band" 5 #ample solution diluted by solvent in
tubing and pump head"
@ 2low rate too high" #olvent flows over
surfae of sorbent 8bands may
disappear down into the sorbent9"
Allow alternating segments of
solvent and air to follow the
sample through the pump"
Radial strea!ing of bands 8same
appearane as broad bands
when rotor is in motion9"
5 *nner edge of the sorbent has
beome impervious by reation with
polar ompounds" *mpervious part
may be visible as a line of dar! spots"
/repurify samples 8page 539"
Remove impervious part of the
sorbent 8page 539"
@ #ample solution ontains unA
dissolved material in suspension"
2ilter or entrifuge sample
solution"
7 #udden inrease in solvent density%
e"g" from he1ane to dihloromethane"
+enser solvent strea!s through the
lighter solvent"
Avoid solvent hanges to
dihloroAmethaneJhloroform or
use a short solvent gradient"
Chromatotron does not separate
sample although separation is
possible on regular T<C plates"
5 Rf is too high" The Rf is sometimes
higher in the Chromatotron than on
regular T<C plates" Common problem
with hloroform A methanol"
-se less polar solvent mi1ture"
@ ($uilibrium onditions in the
Chromatotron versus non e$uilibrium
onditions of regular T<C"
#ee pages 53 and 57 for
operation under non e$uilibrium
onditions"
7 #orbent layer not fully dried before
sraping or air drying to remove
leanup solvent has left moisture in the
layer"
+ry to onstant weight"
+ar! band at outer edge of
sorbent"
Not a problem" #olvent ompletely
fills the sorbent pores at the outer edge
of the layer"
+ar! band at inner edge of
sorbent"
At high flow rates the solvent
ompletely fills the sorbent pores at the
inner edge" Not a problem but an
indiation that flow rate is approahing
ma1imum where solvent flows over
surfae"
<umpy sorbent mi1ture during
pouring" *rregular surfae after
setting" #hort onentri ra!s
may form" #amples will give
nonAirular bands"
5 *nsuffiient water used in reipe"
@ *neffetive mi1ing before pouring"
7 Rapid setting ataly'ed by traes of
hydrated gypsum in the mi1ing 0ar"
Add e1tra @A=K of water"
#ha!e more vigorously" Crea!
up air po!ets with a spatula"
; Mi1ing and pouring of reipe
performed too slowly" Mi1ture has
partly set before pouring"
7>
;40BL25 C"!S2 C!42
#orbent layer slides off or piees
fla!e off from outer edge"
<ayers ra! during drying"
Mi1ed reipe may set before
pouring is omplete"
5 #orbentAgypsum mi1ture was
manufatured with partly hydrated
binder" Common problem with Mer!
silia gel /2"
-se standard grade silia gel"
Che! $uality of binder 8page
@59"
@ *neffetive leaning of rotor before
oating"
An abrasive household leaner
must be used"
Clean with o1idi'ing aid
leaning mi1tures" /lae two
rotors in ontat with the
leaning mi1ture between"
-se oven drying of the sorbent
layer at =3 in plae of air
drying" Oven dried layers are
more firmly bound"
-se glue bound layers 8page
@?9"
7 Cleaned rotor was ontaminated
before oating"
+ry leaned rotors with paper
towels only" +o not dry in an
oven or with aetone" Coat
rotors within 5J@ hr of leaning"
; Mi1ing 0ar ontains hydrated gypsum
from previous pourings"
= Ambient temperature too high" +o not oat rotors when
ambient temperatures are high"
Adhesion is signifiantly wea!er
when the setting ta!es plae
above 73C 84>29"
.ery soft powdery layers" Oven temperature too high" -se =3 ma1"
#raper leaves part of layer
unsraped"
5 Coating performed on sloping benh"
@ Metal dis! of the oating arbor is not
parallel to the rotor due to sorbent
between them"
7 *nsuffiient sorbent A water mi1ture"
The layer is too thin before sraping"
*nrease $uantities in reipe"
#hort ars gouged in the sorbent
during sraping"
2ibers or other foreign material in the
sorbent"
/erformane is not affeted
signifiantly by this defet"
Cirles gouged in the sorbent
during sraping"
#raper blade has rough edge" /erformane is not affeted in
any way"
-. absorbing ompounds not
detetable"
5 Aetone or other -. absorbing
solvent is present in the sample%
sorbent% pump or solvent"
(vaporate leanAup solvents
ompletely"
@ Cen'ene is present as an impurity in
he1ane solvent"
-se ben'ene free grades of
he1ane" -se heptane"
7 -. absorption of hloroform is
mas!ing a wea! hromophore"
-se dihloromethane in plae
of hloroform"
7B
;40BL25 C"!S2 C!42
; Wea! -. lamp" Remove the -. filter from the
lamp"
Add e1tra 'in siliate phosphor
83"=A@K9 8page @39"
= Manufaturer has added insuffiient
phosphor to the sorbent"
Add 3"=A@K of 'in siliate
8page @39"
> #ample si'e too small or
Chromophore is wea!"
#top solvent flow and swith off
Chromatotron" Observe bands
on stationary rotor"
Apply sample as one spot on
rotor 8<ight loading% page 5=9"
-se 5J@ mm layers to maintain
higher onentrations of small
samples"
Condensation on lid" This is normal% not a problem"
Absene of ondensation after 73 min
operation may indiate vapor lea!s"
A slightly fogged lid an be
leared by warming with the
hand" <arger drops will
oalese if the Teflon is tapped"
#ample solution splashes onto
the lid"
2low rate too high during sample
introdution"
+o not e1eed B mlJmin during
sample introdution"
#olvent ba!s up in the
olletion hannel"
5 Outlet blo!ed"
@ /olar solvents do not flow smoothly
through Teflon tubing"
Clear outlet with straightened
small si'e paper lip"
Remove output tube"
2luffy partiles in solvent
olletion hannel" May blo!
output hole"
+ust from rotors stored in the open" Clow off dust before mounting
rotors"
2unnel stopo! develops slow
lea!"
<oose stopo! Remove and s$uee'e stopo!
slightly end to end in a vise"
#olvent flow eases or is
redued to a low rate"
5 #tainless steel filter mesh in funnel
stopo! assembly is logged"
@ RH pump drive belt is loose"
7 Cotton filter logged" Cubbles will
form in the tubing"
; Cotton pa!ed too tightly in the filter"
= .apor lo! in the pump" Common
problem with very volatile solvents at
high ambient temperatures"
> Clo!age in tubing or inlet
Remove stopo! assembly
from funnel and ba!wash"
<oosen the two srews holding
the motor and ad0ust motor
position to tighten the belt"
Replae otton" +o not use
glass wool"
Raise the solvent reservoir
about @3 m to inrease inlet
pressure"
-se less volatile solvents"
Move to a ooler loation"
+isonnet tubing and inlet"
Che! eah part separately"
74
;40BL25 C"!S2 C!42
#olvent lea!s from pump or tube
fittings"
5 *nreased pressure due to blo!age
on output side of pump"
@ 2lared ends of tubing damaged"
+isonnet tubing and inlet"
Che! for blo!ages" +o not
tighten with toolsH Hand tighten
only" Thread tape is not
effetive"
/ump does not start" -nplug pump% remove over
8thumbsrew at rear of RH
pump9"
Ta!e off drive belt% turn pulley
by hand to determine if problem
is in pump head or motor"
Che! belt tension"
#olvent inlet beomes tight in
lid"
5 *nlet has swelled through
absorption of solvents"
@ +eposits on threads"
Allow inlet and lid to dry out for
@; hr"
Clean with solvents or srape
off deposits"
Rethread with =J5>A54 tap and
die"
/artiles of sorbent on Teflon
lid"
5 #orbent dust not blown off after
sraping rotor"
@ (1essive nitrogen flow"
Clow off sorbent after sraping"
Wipe lid with moist paper towel"
Chromatotron osillates from
side to side"
5 Rotor unbalaned due to uneven
evaporation of solvent"
@ -nbalaned home made glass rotor"
(vaporate solvent from rotor
with nitrogen before swithing
off"
Remove rotor and allow solvent
to evaporate"
Rotors ra! at enter" Rotor support ollar and shaft ollar
loose"
#ee Replaing the Rotor
#upport Collar% page 7;"
(ndplate beomes ethed"
2la!y pathes in main vessel"
Corrosion by strong aids% usually
HCl impurity in hloroform"
Add solid sodium biarbonate
or alumina to hloroform" Test
with wet pH paper"
-se dihloromethane in plae
of hloroform"
Chromatotron produes a
rubbing sound that ontinues
after swithing off% until the
instant the rotor stops"
+eposits on felt seal ausing adhesion
to the rotor support ollar"
No maintenane re$uired
unless adhesion prevents start
up of motor" #ee the 2elt #eal%
page 7;"
Motor does not start" Rotor support ollar in ontat with
main vessel"
#ee Ad0usting the Main .essel%
page 7;"
(lino motor has insuffiient
tor$ue" #low start up" May run
at half speed"
<ow supply voltage" -se motor apaitor 5 M2+
larger than speified or
inrease supply voltage with
variable transformer"
#hoehorn or endplate !ey lost" -se a small he1 !ey 8Allen
wrenh9"
7?
::::::::::::::::::C3405"T0T401 ;"4TS LIST
/art Number +esription
4?@;A37 *nstrution manual for model 4?@; Chromatotron
HA3> )lass rotor
HA3B3 #raping tool
HA3B5 #raper blade% 5 mm
HA3B@ #raper blade% @ mm
HA3B; #raper blade% ; mm
HA3BAD #raper blade% D mm" ")ive re$uired D in range 3"7 to ;"3
HA3B? 2inishing sraper blade
HA5> Coating arbor
HA5? Collar endplate 8with Teflon washer9
HA@; Teflon washer for ollar endplate
HA7; ,ey for endplate
HA7> Output tube
HA;3 #hoe horn
HA;4 <id retaining lips% set of 4
HA=7 /ump onnetions !it for RH% R/) and Q) pumps"
2ilterJstopperJtubingJinlet
HA=7A7 Coupling
HA=; #olvent filter
HA== #olvent pump model RH" *nludes pump onnetions !it HA=7
HA>= Wi! and @ wire wi! holders
HA>B #olvent inlet for use with pump
HA>4 #olvent inlet for diret introdution of sample 8diagram below9
HA?3 )ilmont flowmeter% 53A@533 mlJmin% for nitrogen
HA?@ Teflon lid with solvent inlet for use with pump
HA?7 Replaement Teflon sheet for Teflon lid
HA?? /olyethylene over
HA53> @;. power supply
;3
CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC";;216IH
T32 T2ST 5IHT!42
A mi1ture of high Rf olored ompounds is useful for testing sorbent layers"
FThe test mi1tureF refers to a mi1ture of the @%;Adinitrophenylhydra'ones of
ylopentanone and yloheptanone" The same derivatives of openAhain
!etones are also suitable" A'oben'ene derivatives and the ommerial dye
mi1tures used with regular T<C plates are not reommended sine they are
not ompletely stable and ontain low Rf impurities that remain on the sorbent"
;5
CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCI162H
adsorbent #ee sorbent
air drying of layers @B
aluminum o1ide layers @7
band slope 7@
bands
broad 7=% 7>
eentri 7=
irregular 7=
strea!ing 7>
binders @3
hanging
rotors ?
wi! =% >
hromatography
partition 75
rapid 5;
reversed phase 73
Chromatotron
diagram @
installation ;
introdution 5
parts list ;3
setAup diagram 7
oating rotors
ellulose layers @;
introdution @3
glueAbound layers @?
gypsumAbound layers @;
leaning rotors @=
leanAup of sorbent layers 5B
olletion of frations 5>
onnetion in series 5?
detetion
small samples 5=
-. absorbing samples 5=
-. transparent samples 5>
development% multiple 54
eletrial onnetions 77
felt seal 7;
fration olletion 5>
fration olletors 5B
glueAbound layers @?
gradient elution 55
inlet =
insoluble samples 55% 57
layer thi!ness ?
layers
fla!ing 7B
loose 7B
soft 7B
less soluble samples 55% 57
main vessel ;
ad0usting 7;
maintenane and repairs 77
ma1imum flow rate 5;
mi1ing 0ar @5% @=
multiple development 54
nitrogen flow 4
oven drying @B
partition hromatography 75
parts list ;3
phosphors @3
polyethylene glyol @3% @7
prepurifiation of samples 53
pumps >A4% 77% 74% 7?
rapid hromatography 5;
reipes
aidified silia gel A gypsum @@
aluminum o1ide @7
mi1ing and pouring @=
silia gel A glue @;
silia gel A polyethylene
glyol A gypsum @7
silia gel A silver nitrate A
gypsum @7
reyle 54
Rf 53
rotor support ollar 7;
rotors
hanging ?
;@
leaning @=
oating @3A7@
glueAbound layers @?
gypsumAbound layers @;
partition layers 75
setAup for slow setting
layers 75
storage @?
sample
introduing and eluting 55% 5@% 57
loading 5=
low solubility 55% 57
prepurifiation 53
volume 57
sraping sorbent layers @4
series onnetion 5?
silia A gel glue layers @;% @?
silia gel A gypsum layers @5 A @7
solvent
addition 55
hoie 53
ondensation 5@% 74
flow rates 55% 5;
ma1imum 5;
inlet =
interrupting flow 5=
lea!s 74% 7?
pumps >A4% 77% 74% 7?
surfae flow 5;
solvent inlet =
sorbent
leanAup 5B
layer thi!ness ?
loose layers 7B
sorbents
8list9 @3
oating rotors @3
drying layers @>
sraping layers @4
storage of oated rotors @?
surfae flow 5;
tailing 53
Teflon lid ;% 7;
test mi1ture ;5
Troubleshooting 7=
vapor lo!s 5@% 74
wi! =% >
;7