Current Protocols in Nucleic Acid Chemistry || Thin-Layer Chromatography

APPENDIX 3DThin-Layer ChromatographyC.L.F. Meyers1 and D.J. Meyers1

1Johns Hopkins University School of Medicine, Baltimore, Maryland

ABSTRACT

TLC is used extensively in nucleic acid chemistry to monitor the progress of chemicalreactions, to assay fractions collected from a larger chromatographic separation (e.g.,column chromatography), and to determine optimal conditions prior to column chro-matography. This unit describes methods for spotting test compounds onto a TLC plate,developing the plate in a suitable solvent system, visualizing the results, and calculatingthe retention factor (Rf). Candidate compounds can be co-spotted for identification with-out relying on Rf values. Curr. Protoc. Nucleic Acid Chem. 34:A.3D.1-A.3D.13. C© 2008by John Wiley & Sons, Inc.

Keywords: thin-layer chromatography � TLC � nucleosides � nucleotides � separation

INTRODUCTION

Thin-layer chromatography (TLC) is used as a tool for determining compound identityand purity and for quantitation. Most commonly, this technique is used to monitor theprogress of a chemical reaction or to assay fractions collected from a larger chromato-graphic separation (e.g., column chromatography). TLC is typically performed usingpaper or commercially available glass, aluminum, or plastic plates that are coated witha thin layer of adsorbent (TLC plates). Commonly used adsorbents for separation of nu-cleic acids include cellulose, DEAE-cellulose, PEI-cellulose, silica gel, or C18 reversed-phase adsorbents. For example, silica gel is a polar adsorbent commonly used to separatecompounds of low to medium polarity. Highly polar compounds will “stick” to polaradsorbents and may be purified instead using C18 reversed-phase TLC plates (glassplates coated with a highly lipophilic adsorbent). In reversed-phase separations, nonpo-lar compounds will “stick” to the adsorbent, making it possible to separate highly polarcompounds. Regardless of the adsorbent used, TLC can be used to run test separationsin preparation for column chromatography.

This unit describes a method for spotting a test compound or reaction mixture onto aTLC plate and developing the plate in a suitable solvent system, as well as the variousmethods for visualizing the results, which are then used to calculate retention factor (Rf)values (see the Basic Protocol). The Alternate Protocol describes co-spotting the platewith candidate compounds in order to identify the unknown sample without relying onRf values. Instructions for cutting TLC plates are given in Support Protocol 1, and aprocedure for preparing spotters is given in Support Protocol 2.

STRATEGIC PLANNING

Adsorbent and Eluent

Several possible combinations of adsorbent and eluent exist for TLC separation of nu-cleosides and nucleotides. A few TLC conditions are broadly useful for resolving bothnucleosides and nucleotides. In general, mixtures of polar and nonpolar organic sol-vents, alcohol/water/acid, alcohol/water/base, or buffered eluents can be used to separatenucleic acids of all types on silica gel, cellulose, or C18 reversed-phase adsorbents(Table A.3D.1, adapted from Steinberg et al., 1996). Resolving neutral nucleosides or

Current Protocols in Nucleic Acid Chemistry A.3D.1-A.3D.13, September 2008Published online September 2008 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142700.nca03ds34Copyright C© 2008 John Wiley & Sons, Inc.

Commonly UsedTechniques

A.3D.1

Supplement 34

Thin-LayerChromatography

A.3D.2

Supplement 34 Current Protocols in Nucleic Acid Chemistry

Table A.3D.1 Examples of Adsorbent/Eluent Combinations for Separation of Nucleic Acidsa

For separation of

Adsorbent Eluent Nucleosides Nucleotides

Microcrystalline iPrOH/HCl/water (65:16.7:18.3 v/v) Y Y

cellulose (Avicel) Saturated (NH4)2SO4/1 M NaOAc/iPrOH (80:18:2) Y Y

Isobutyric acid/water/25% NH4OH (400:208:0.4) Y N

iPrOH/water/conc. NH4OH (85:15:1.3) Y N

n-Butanol/water/formic acid (77:13:10) Y N

DEAE-celluloseb Isobutyric acid (conc.)/1 M NaOAc/iPrOH (80:18:2) Y Y

0.005 N HCl Y Y

Isobutyric acid (conc.)/NH4OH/water (33:1:16) Y Y

Saturated (NH4)2SO4/1 M NaOAc/iPrOH (40:9:1) Y N

PEI-celluloseb LiCl (0.25-1.6 M) N Y

Formic acid/sodium formate, pH 3.4 (0.5-4.0 M) N Y

1.0 N formic acid N Y

2.0 N formic acid/LiCl (1:1, 0.5-2 M) N Y

Silica gel Combinations of polar and non-polar solvents. Polarsolvents include MeOH, EtOH, EtOAc, and acetone.Non-polar solvents include CHCl3, CH2Cl2, hexanes,petroleum ether, and toluene.

Yc Yc

aAdapted from Steinberg et al. (1996).bDEAE-cellulose, diethylaminoethyl cellulose; PEI-cellulose, poly(ethyleneimine)-HCl prepared cellulose.cFor protected or masked nucleosides or nucleotides.

protected nucleotides is often accomplished on silica gel TLC plates using combinationsof polar and nonpolar organic solvents (polar solvents include methanol, ethanol, ethylacetate, and less polar solvents include chloroform, methylene chloride, and hexanes).Highly polar solvents (i.e., water/alcohol) are required to resolve ionic nucleotides onsilica gel, as silica gel is itself highly polar and retains polar compounds. Nucleoside phos-phoramidites used in oligonucleotide synthesis are basic and may “streak” on an acidicsilica gel TLC plate. To overcome this problem, 1% ammonia or triethylamine is oftenadded to the eluent to “deactivate” the silica gel. Likewise, acid-sensitive dimethoxytritylprotecting groups often used for protection of nucleoside hydroxyl groups may be stabi-lized on a silica gel TLC plate by addition of a base such as triethylamine. A compre-hensive summary of TLC conditions used for separation of nucleic acids can be foundin Steinberg et al. (1996).

Visualization Method

Only colored compounds may be visualized by the naked eye following development ofthe TLC plate; as many compounds are not colored, an alternative method for visualizingthe plate is necessary. There are numerous methods for visualizing TLC plates (seeZweig and Sherma, 1972 for more information). Methods for visualization fall into threeclasses: destructive, semi-destructive, and nondestructive.

A variety of stains and dips fall into the class of destructive visualization methods, becauseany compound present on the plate will decompose to give a dark spot. Some commonvisualization agents used in synthetic organic chemistry are summarized in Table A.3D.2.Several of these agents may be considered “universal” or “general” stains since they


A.3D.3

Current Protocols in Nucleic Acid Chemistry Supplement 34

Tab

leA

.3D

.2Ty

pica

lSta

ins

Use

din

Org

anic

Che

mis

try

for

Vis

ualiz

atio

nof

Spo

tson

TLC

Stai

nTy

pica

lcol

or(s

)U

seR

ecip

e

p-A

nisa

ldeh

yde

(p-

met

hoxy

benz

alde

hyde

)V

ario

usco

lors

ona

light

pink

back

grou

nd

Gen

eral

aSl

owly

add

3.33

mL

conc

.sul

furi

cac

idto

90m

Let

hano

lcoo

led

inan

ice/

wat

erba

th.O

nce

addi

tion

isco

mpl

ete,

rem

ove

from

ice/

wat

erba

than

dad

d1

mL

acet

icac

idan

d2.

5m

Lp-

anis

alde

hyde

.

Bro

moc

reso

lgre

enY

ello

wsp

ots

ona

blue

back

grou

ndO

rgan

icac

ids

(pH

<5)

Dis

solv

e0.

04g

brom

ocre

solg

reen

in10

0m

Let

hano

l;ad

ddr

opw

ise

a0.

1M

solu

tion

ofso

dium

hydr

oxid

eun

tilso

lutio

ntu

rns

pale

blue

Cer

ium

amm

oniu

mm

olyb

date

(CA

M,

Han

essi

an’s

stai

n)

Var

ious

colo

rson

light

blue

orgr

een

back

grou

nd

Gen

eral

aTo

235

mL

wat

er,a

dd12

gam

mon

ium

mol

ybda

tete

trah

ydra

te[(

NH

4) 6

Mo 7

O24

·4H2O

],0.

5g

amm

oniu

mce

rium

(IV

)su

lfat

edi

hydr

ate

[Ce(

NH

4) 4

(SO

4)·2

H2O

],an

d15

mL

conc

.sul

furi

cac

idTo

450

mL

wat

er,a

dd25

gam

mon

ium

mol

ybda

tete

trah

ydra

te,5

gam

mon

ium

ceri

um(I

V)

sulf

ate

dihy

drat

e,an

d50

mL

conc

.sul

furi

cac

id

To36

0m

Lw

ater

,add

10g

amm

oniu

mm

olyb

date

tetr

ahyd

rate

,4g

amm

oniu

mce

rium

(IV

)su

lfat

edi

hydr

ate,

and

40m

Lco

nc.s

ulfu

ric

acid

Nin

hydr

inSh

ades

ofpu

rple

orbr

own

Am

ino

acid

san

dam

ines

0.3

gni

nhyd

rin,

100

mL

n-bu

tano

l,an

d3

mL

glac

iala

cetic

acid

1.5

gni

nhyd

rin,

500

mL

95%

etha

nol,

and

5m

Lgl

acia

lace

ticac

id

Phos

phom

olyb

dic

acid

Gre

ensp

ots

onlig

htgr

een

back

grou

nd

Gen

eral

bD

isso

lve

10g

phos

phom

olyb

dic

acid

in10

0m

Lab

solu

teet

hano

l

Pota

ssiu

mpe

rman

gana

teY

ello

wsp

ots

ona

purp

leba

ckgr

ound

Use

fulf

orox

idiz

able

func

tiona

lgr

oups

In30

0m

Lw

ater

,dis

solv

e3

gpo

tass

ium

perm

anga

nate

(KM

nO4),

20g

pota

ssiu

mca

rbon

ate

(K2C

O3),

and

add

5m

Lof

5%(w

/v)

sodi

umhy

drox

ide

(NaO

Hin

wat

er)

Van

illin

Var

ious

colo

rsG

ener

ala

Add

6g

vani

llin

to10

0m

Lof

95%

etha

nola

ndad

d1

mL

conc

.sul

furi

cac

ida

Phot

osen

sitiv

e;pr

otec

tfro

mlig

ht.

bTe

mpe

ratu

rese

nsiti

ve;s

tore

refr

iger

ated

.


A.3D.4


allow visualization of many different functional groups or classes of compounds. Otherstains may only work well for a certain class of compounds (i.e., glycosides or lipids).Touchstone (1992) lists over 200 stains that can be used for different functional groups(see chapter on Visualization Procedures therein). Recipes for many stains can also befound online (see Internet Resources). Several stains are particularly noteworthy fornucleosides and nucleic acids. p-Anisaldehyde in sulfuric acid (Table A.3D.2) stainssugars, and is thus commonly used to detect nucleosides (e.g., UNIT 1.3). In addition,acidic solutions can be used to detect the trityl group as a bright orange spot. Examplesof stains used to visualize the trityl group include 10% to 20% sulfuric acid (UNITS

4.14 & 5.3); hydrochloric acid (UNIT 2.8); 3:2 (v/v) 60% perchloric acid in ethanol (UNIT

4.27); and 3% trifluoroacetic acid in dichloromethane (UNIT 1.5). Ninhydrin can be usedto detect amino-modified nucleosides (Table A.3D.2; UNIT 4.9). Finally, 2,6-dibromo-4-benzoquinone-N-chloroimine (DBPNC; 2.5 mg/mL in ethanol) is used to detect sulfur-containing compounds such as phosphorothioates, which are visualized as pink spots(UNIT 4.9).

Iodine vapors are commonly used as a semi-destructive visualization method. Manycompounds will absorb iodine vapors and turn dark brown on a lighter brown background.Iodine has a strong affinity for unsaturated or aromatic compounds and can act as anelectrophile to add across double bonds or with nucleophilic amines. The color changeis not always permanent, however, so spots should be marked with a pencil for futurereference.

UV light can be used to visualize certain compounds on commercial TLC plates that con-tain a short-wavelength (254 nm) or long-wavelength (366 nm) UV indicator. Compoundscontaining a UV-absorbing chromophore are termed UV-active and can be visualized us-ing this method. Such compounds block the absorbance of the UV light by the fluorescentindicator, creating a dark spot against a bright background. For this reason, the methodis often referred to as UV shadowing. UV shadowing is termed nondestructive becausenonphotolabile compounds generally do not decompose under these conditions. Virtu-ally all nucleosides absorb light at 254 nm and can be visualized at this wavelength.Some nucleoside analogs absorb at 366 nm and are best visualized with plates containingthe longer-wavelength fluorophore. Hand-held UV lamps with both long- and short-wavelength UV light are commercially available for this purpose. Visible dark spotsshould be circled with a pencil for future reference.

BASICPROTOCOL

THIN-LAYER CHROMATOGRAPHY

The preparation of a TLC plate is accomplished by dissolving the mixture of compoundsto be separated in a suitable solvent (e.g., acetone, chloroform, ethyl acetate), spottingthe mixture onto a TLC plate, and allowing the solvent to evaporate. The TLC plate isthen developed in a TLC chamber (Fig. A.3D.1) containing a suitable solvent system.The solvent in the chamber is drawn upward onto the plate by capillary action, carryingalong with it the compounds present in the mixture. Separation of compounds occursbecause each compound is retained differently on the adsorbent. When the solvent haseluted to the top of the plate, the TLC plate is removed from the chamber. Followingvisualization, the retention factor, or Rf value, of each spot is determined. The Rf valuemeasures how strongly each compound is retained on the adsorbent in a particular solventsystem. Compounds with large Rf values migrate higher up the plate than compoundswith small Rf values.


A.3D.5


baseline above the level of the solvent

solventsystem

solventfront

cover

Figure A.3D.1 Diagram of a TLC developing chamber.

Materials

Test compoundAppropriate volatile solvent for test compoundAppropriate solvent system (eluent; see Strategic Planning)Reagent(s) for visualization (stain; see Strategic Planning)Solid iodine (for semi-destructive visualization)

TLC plate with appropriate adsorbent (see Strategic Planning), cut to size (seeSupport Protocol 1)

TLC spotter (see Support Protocol 2)Wide-mouthed TLC chamber or beaker with lid (Fig. A.3D.1)Filter paper100◦ to 140◦C heat gun or hot plate (for destructive visualization)Hand-held UV light source, 254 or 366 nm (for nondestructive visualization)

Spot TLC plate1. Using a pencil, draw a faint line at the edge of an appropriately sized TLC plate,

∼1 cm from the bottom of the plate (Fig. A.3D.1).

This is the baseline of the TLC plate. If spotting more than one compound along thebottom of the plate, it may be helpful to make the appropriate number of notches on thebaseline to mark the position of each spot (Fig. A.3D.2). Generally, spots should be spaced∼5 mm from each other and from each edge of the plate.

IMPORTANT NOTE: Mark the plate very faintly, being sure not to score the layer ofadsorbent. Drawing heavily on the TLC plate will cause the adsorbent to be scratchedaway.


A.3D.6


spot the TLC plate

mark the baseline

faintly “notch” the position of each spot

~ 1 cm

~ 5 cm

spotted mixture to be analyzed

Figure A.3D.2 Spotting a TLC plate. The baseline is ∼1 cm from the bottom and spots are∼5 mm apart.

2. Dissolve the mixture of compounds to be analyzed in a volatile solvent (e.g., acetone,ethyl acetate, dichloromethane, or aqueous mixtures, if necessary).

If using a C18 reversed-phase TLC plate, the compound may dissolve only in wa-ter/organic solvent mixtures. Dissolve the compound in as little water as possible.

3. Draw a small amount of the dissolved mixture up into the tip of a TLC spotter andbriefly touch the tip of the spotter onto the baseline of the TLC plate in the appropriateposition to deposit a small amount of sample in the form of a spot. Make the spot assmall as possible (typically ≤3 mm). Let the solvent in the spot evaporate.

The longer the tip of the spotter is held on the plate, the larger the spot will be.

4. Repeat along the baseline of the TLC plate until the desired number of mixtures tobe analyzed has been spotted (Fig. A.3D.2).

Do not spot mixtures too closely together along the baseline or too close to the edge (seestep 1). Overlapping spots will “bleed” into each other’s path of migration, making itdifficult to identify the origin of each spot. Edge effects can also interfere with migration,especially with water-containing eluents.

5. Repeat the spotting of each mixture in the same place on the TLC plate in order toaccumulate a sufficient amount of each compound at the baseline of the plate.

Be careful not to make the spots too concentrated, as mixtures of compounds that have beenoverloaded onto the TLC plate will appear “streaky” and unresolved when developed.See Figure 6.1 in Touchstone (1992) for an example of the effect of sample load on theresolution of a mixture.

Compounds dissolved in water/organic solvent mixtures will take longer to dry.

If the compound absorbs UV light, the plate can be visualized by UV absorption todetermine whether enough compound has been loaded onto the plate (see step 9c).

Develop plate6. Choose an appropriate solvent system (eluent) to develop the TLC plate. Pour the

chosen eluent into a wide-mouthed TLC chamber or beaker to a depth of no more


A.3D.7


than 5 mm, and cover the chamber to prevent evaporation. If necessary, line the insideedges of the chamber with filter paper in order to saturate the chamber atmospherewith the eluent vapors and prevent solvent evaporation.

Choosing an eluent is often more complicated than it sounds. Each separation carriedout on a TLC plate will require a different solvent system, which will depend uponthe adsorbent chosen for the separation. In general, organic and/or aqueous solventsystems are used for separations carried out on silica gel, while water/acetonitrile,water/alcohol, water/alcohol/acid, water/alcohol/base, or buffered eluents are used forseparations carried out on cellulose and C18 reversed-phase medium. Refer to the CRCHandbook of Chromatography (Zweig and Sherma, 1972) and the Handbook of Thin-Layer Chromatography (Sherma and Fried, 1996) for guidance in choosing a solventsystem for a given separation.

7. Place the TLC plate into the chamber, making sure the baseline is above the level ofthe eluent in the chamber. Cover the chamber and allow the solvent to travel upwardon the TLC plate (Fig. A.3D.1).

Development of silica gel plates may take only a few minutes, whereas the developmentof cellulose or C18 reversed-phase TLC plates may take considerably longer.

Aqueous solvent systems elute up the TLC plate much more slowly than organic solventsystems. For example, elution of an aqueous mobile phase up a 10-cm TLC plate maytake ∼45 min.

8. When the eluent nears the top of the plate, remove the plate from the chamber,mark the position of the solvent front, and allow the eluent to evaporate in a fumehood.

CAUTION: Do not breathe solvent vapors.

C18 reversed-phase TLC plates developed with aqueous solvent systems will take severalminutes to dry.

Visualize TLC plate9a. Destructive visualization: Spray the visualization agent onto the TLC plate or dip

the plate directly into the visualization agent. After dipping, remove excess stain byblotting the edges with a paper towel. Record the appearance of any colored or darkspots (some compounds in certain reagents will stain immediately without heating).Next, slowly heat the plate in a chemical fume hood with a heat gun or hot plate(100◦ to 140◦C is appropriate for most stains), and again record the appearance ofany colored or dark spots.

CAUTION: Comparison of spot intensity is not ideal for quantitation because manystains, including the general stains listed in Table A3.D.2, may give a false sense of therelative amounts based on stain intensity.

IMPORTANT NOTE: Do not heat the TLC plate too long or at too high a temperature.For many reagents, excessive heating times or temperatures will cause the backgroundcolor to intensify, making it difficult to visualize spots.

9b. Semi-destructive visualization: Assemble a wide-mouthed, covered chamber con-taining a few crystals of iodine. Place the TLC plate inside the chamber and letit stand for several minutes. Remove the plate and circle the colored spots with apencil.

Most, but not all, compounds present on the plate will absorb iodine vapors and changecolor. Over time, the spots will disappear.

9c. Nondestructive visualization: If using TLC plates containing a short-wavelength orlong-wavelength UV indicator, shine short-wavelength or long-wavelength UV light


A.3D.8


baseline

solvent front

A B C

Figure A.3D.3 Results of TLC. (A) The spot has a Rf of 0.0. For silica gel adsorbent, this meansthat the eluent is not polar enough. For C18 reversed-phase adsorbent, this means that the eluentis too polar (water content too high). (B) The spot has a Rf of 0.94. For silica gel adsorbent, thismeans that the eluent is too polar. For C18 reversed-phase adsorbent, this means that the eluentis not polar enough (organic content too high). (C) The mixture contains two compounds, as isapparent when the TLC plate is developed using a suitable eluent. The spots have Rf values of0.29 and 0.67.

on the plate to produce dark spots against a green or blue background, respectively.Circle visible dark spots with a pencil.

CAUTION: UV light is damaging to the eyes and exposed skin. Protective eyewear shouldbe worn at all times while using a UV light source.

Occasionally, a compound may exhibit fluorescence under UV light and will glow on theTLC plate.

Determine Rf value10. Measure the distance from the baseline to the center of each spot to determine the

distance the spot traveled on the TLC plate.

11. Measure the distance from the baseline to the solvent front to determine the distancethe solvent traveled on the TLC plate.

12. Divide the distance the spot traveled by the distance the solvent traveled to obtainthe Rf value, which ranges from 0.0 to 1.0.

Values <0.2 or >0.8 are not particularly informative. These spots may contain mixturesof compounds that were not separated during the development of the plate (Fig. A.3D.3).Spots with Rf values <0.2 or >0.8 should be reevaluated by adjusting the polarity of theeluent to obtain Rf values within the range of 0.2 to 0.8.

ALTERNATEPROTOCOL

CO-SPOTTING A TLC PLATE

Although the Rf value of a particular spot may give some information about the identityof a compound, it is not always exactly reproducible. For example, an Rf value mayvary depending upon the amount of sample spotted on the plate. For this reason, anRf value alone cannot be used as a certain indication of compound identity. However,the identity of a spot can be determined by co-spotting the unknown compound with


A.3D.9


develop the TLC plate

solvent front

baseline43214321

one spot,no separation

Figure A.3D.4 Co-spotting a TLC plate (see Alternate Protocol). Lane (1) unknown compound.Lane (2) authentic compound A. Lane (3) authentic compound B. Lane (4) mixture of authenticcompounds A and B and unknown compound. Notice that the unknown (lane 1) and authenticcompound B (lane 3) have similar Rf values (0.44 and 0.40, respectively) when run side-by-sideon the TLC plate. Upon closer examination of lane 4, it is shown that, in fact, the unknown andauthentic compound B are identical.

standards of the “authentic” compound (i.e., the compound the unknown is suspected tobe). In this protocol, a method for determining the identity of an unknown compound aseither authentic compound A or B is described (Fig. A.3D.4).

Additional Materials (also see Basic Protocol)

Unknown compoundAuthentic compounds A and B2 × 5–cm TLC plate (see Support Protocol 1)

1. Cut a TLC plate that is wide enough to hold four spots at the baseline (one spot every5 mm; see Support Protocol 1).

The first three positions will be used to spot each compound by itself. The fourth positionwill be used to spot all three compounds together.

2. Spot the unknown compound at the first and fourth positions on the TLC plate. Allowthe solvent to evaporate completely.

3. Spot authentic compound A in the second and fourth positions. Again, allow thesolvent to evaporate completely.

4. Spot authentic compound B at the third and fourth positions.

The TLC plate should be spotted as in Figure A.3D.4.

5. Develop the TLC plate using a suitable eluent chosen for the unknown (see BasicProtocol, steps 6 to 8).

6. Visualize the developed TLC plate (see Basic Protocol, steps 9 to 12).

The TLC plate should demonstrate the results shown in Figure A.3D.4. Notice that theunknown and authentic compound B have similar Rf values when run side-by-side on theTLC plate. Upon closer examination of the fourth position, the lack of separation betweenthe unknown and compound B confirms that they are identical.


A.3D.10


SUPPORTPROTOCOL 1

CUTTING TLC PLATES

Precoated glass TLC plates (10 × 20 cm) are commercially available with a variety ofadsorbents. Before performing TLC, the larger glass plates need to be cut down to a work-able size. Prescored TLC plates are commercially available and can simply be broken atthe right positions. Alternatively, cutting can be done manually using a cutting tool, as des-cribed here. Note that plates with a plastic or aluminum backing can be cut with scissors.

This protocol produces approximately twenty 2 × 5–cm plates. Longer plates may beneeded for applications with longer elution times, which are generally required for moredifficult separations. The length of the plate can be adjusted as needed to achieve thedesired separation. The width of the plate can be adjusted to accommodate the numberof spots that are to be loaded and the size of the chamber used to develop the plate.

Materials

10 × 20–cm TLC platesFilter paper or paper towelRulerGlass cutter (diamond cutters preferred) or scissors

1. Place a 10 × 20–cm TLC plate adsorbent-side-down on a large piece of filter paperor paper towel.

Do not use printed paper as it contains dyes and chemicals that may be transferred to theadsorbent on the plate.

When handling precoated TLC plates, always wear gloves or be sure to handle with cleanhands. Dirt or oil from the hands can be transferred to the adsorbent.

2. To guide the cut, hold a ruler down the center of the plate lengthwise and score theplate using a glass cutter (diamond cutters work best). Break the plate along thescore, producing two ∼5 × 20–cm plates.

CAUTION: Cut TLC plates are very sharp.

C18 reversed-phase TLC plates may “flake” when broken along the scored line.

3. Place each plate adsorbent-side-down on the filter paper. Holding the ruler as a guide,make a series of scores across each plate, ∼2 cm apart. Break the plates along thescores to make approximately twenty 2 × 5–cm TLC plates.

TLC plates of this size should be appropriate for three spots at 5 mm spacing. Withexperience, for routine monitoring of column chromatography, it may be possible to spotup to six or seven very small spots at smaller intervals.

SUPPORTPROTOCOL 2

PREPARATION OF TLC SPOTTERS

The spotter is the apparatus used to load the mixture of compounds onto the TLC plate.It is appropriately named because it makes a “spot” on the plate. Spotters are easilyprepared from capillary tubes, as described here. Alternatively, commercially available,disposable micropipets can be used as spotters. These are sold in packages of 100 andare available from most scientific supply companies (e.g., 1 μL; Drummond).

Materials

∼1 × 100–mm open-ended capillary tubesBunsen burner

1. Put the center of an open-ended capillary tube into a small blue Bunsen burner flame.Hold it in the flame until the center of the capillary tube begins to liquefy.

Do not leave the capillary tube in the flame too long or it will melt the edges of the tubetogether in the center.


A.3D.11


Figure A.3D.5 Preparation of TLC spotters.

2. Immediately remove the capillary tube from the flame and pull the ends in oppositedirections (Fig. A.3D.5).

3. Break the capillary tube in the center to make two spotters.

Several spotters may be needed for loading different compounds onto the TLC plate.

It is good practice to use a different spotter for each sample; however, in some cases itmay be necessary only to “wash” the spotter before using it again (e.g., for analyzingfractions collected during column chromatography). To wash a spotter, simply draw asuitable solvent (e.g., acetone) up into the thin end (tip) of the spotter. Release the acetoneonto a paper towel by touching the tip of the spotter to the towel. Repeat several times toensure that all of the previous sample has been removed.

COMMENTARY

Background InformationThin-layer chromatography (TLC) has

emerged as a highly flexible and indispensabletool for the identification and quantificationof organic molecules, including nucleic acids.While gel electrophoresis techniques have ob-viated the need for TLC of large oligonu-cleotides, TLC remains an important tool for

analysis of small nucleotides and nucleosides.Together with high-performance liquid chro-matography (HPLC), TLC is primarily utilizedfor preliminary compound characterization,for monitoring the progress of a chemical re-action, and for identifying compounds elutedfrom a larger chromatographic separation. Forthe analysis of nucleotides and nucleosides,


A.3D.12


TLC takes advantage of the differences in po-larity or ionization state to facilitate compoundseparation on a stationary phase (or adsor-bent). In practice, this method is simple andinexpensive and provides rapid informationabout the number of components in a mixture.TLC may also serve as a method for determi-nation or confirmation of compound identityby comparison to known compounds. The ad-vantages of this technique and its many appli-cations are reviewed in detail in Sherma andFried (1996).

Critical Parameters andTroubleshooting

The combination of adsorbent and eluent iscritical to the success of TLC. Some knowl-edge of the structures of possible componentsin the mixture to be analyzed is helpful fordetermining the appropriate combination ofadsorbent and eluent. For example, if a mix-ture of nucleotides is being analyzed, ion-exchange conditions (DEAE-cellulose usingbuffered eluent) should be considered for TLC,since this class of compounds bear chargedphosphate moieties that are distinguishable byion-exchange chromatography.

Visualization of spots on the TLC plate isalso critical to the success of TLC. Again, it ishelpful to have some knowledge of compoundstructures in order to determine the best visu-alization method. If this is not possible, de-structive visualization may be used generally.

Common problems encountered duringchromatographic separations by TLC includecompound migration with the solvent front(Rf > 0.8), compound adsorption at the base-line (Rf < 0.2), and compound “streaking” dur-ing migration up the TLC plate. Rf values thatare < 0.2 or > 0.8 are not particularly informa-tive. Spots with very low or very high Rf valuesmay be composed of components that were notseparated during development of the plate andmay give misleading results. The appropriateselection of the adsorbent/eluent combinationis essential to prevent this outcome. Adjust-ment of eluent polarity often addresses thisproblem for separations carried out on silicagel or C18 reversed-phase TLC plates. For ex-ample, increasing the eluent polarity will in-crease Rf values of polar compounds on silicagel, while increasing eluent polarity will de-crease Rf values of nonpolar compounds onC18 reversed-phase TLC. Likewise, increas-ing ionic strength of the eluent in ion-exchangeTLC may result in faster migration (higher Rf)of ionic compounds on the TLC plate. In addi-

tion, compounds may “streak” during migra-tion up the TLC plate and result in overlappingof migrating components and inefficient sepa-ration. Streaking may result from overloadingof the sample onto the TLC plate, degradationof components (often observed on silica gel),or protonation of basic amines on acidic silicagel. This problem is often solved by spotting asmaller sample onto the TLC plate, or by theaddition of a small percentage of a highly po-lar solvent, such as methanol or ethanol to theeluent. Compound degradation or protonationof basic amines may be avoided by deactivat-ing silica gel by the addition of 1% (v/v) 7 Nammonia in MeOH (commercially available)or 1% triethylamine to the eluent.

Anticipated ResultsTLC of nucleosides and nucleotides results

in the resolution of complex mixtures and canprovide information about the identity of com-ponents in a mixture. The results of TLC areused to purify compounds either from the TLCplate itself or from a larger chromatographicseparation.

Time ConsiderationsTLC can be performed rapidly; however,

some time may be required to identify an ap-propriate eluent. Depending upon the com-plexity of the mixture being analyzed, op-timization of the eluent may require severalminutes to several hours, as numerous elutionconditions are often attempted to achieve asuitable separation. Once all materials are as-sembled and an appropriate eluent has beenidentified, carrying out the TLC analysis re-quires several minutes for eluents containingvolatile solvents, and up to 1 to 2 hr for elu-ents containing aqueous mixtures. The time todevelop a TLC plate is also dependent uponthe length of the plate. For example, a 10-cmTLC plate may be developed in ∼45 min us-ing an aqueous eluent. Longer times may berequired for more difficult separations. Fol-lowing development of a TLC plate, visual-ization is generally achieved in a few minutes,depending upon the visualization method.

Literature CitedSherma, J. and Fried, B. (eds.) 1996. Chromato-

graphic Science Series, Vol. 71: Handbook ofThin-Layer Chromatography. Marcel Dekker,New York.

Steinberg, J., Cajigas, A., and Oliver, G. 1996.Nucleic acids and their derivatives. In Chro-matographic Science Series, Vol. 71: Handbookof Thin-Layer Chromatography (J. Sherma and


A.3D.13


B. Fried, eds.) pp. 921-969. Marcel Dekker,New York.

Touchstone, J. 1992. Practice of Thin Layer Chro-matography, 3rd ed. John Wiley & Sons,New York.

Zweig, G. and Sherma, J. 1972. CRC Handbook ofChromatography, Volumes I and II. CRC Press,Cleveland, Ohio.

Internet Resourceshttp://www.ux1.eiu.edu/∼cfthb/research/handbook/

TLCstains.htmExcellent reference for TLC stain recipes, the colorsthe stain produces, and functional groups sensitiveto that particular stain.

http://www.chemicalshift.com/chemistry/chemistry-tips/tlc-stains/

This site contains TLC stain recipes.

http://chem.chem.rochester.edu/∼nvd/tlcnotes.html

Good reference for practical TLC use. Links to or-ganic synthesis tips.

http://chem.ps.uci.edu/∼srychnov/tlc stain recipes.htm

This site is another good source containing TLCstain recipes.

http://www.umich.edu/∼mssgroup/Group%20Business/TLCStains.pdf

This site contains information on TLC stains andtheir uses.

http://stoltz.caltech.edu/files/Techniques/TLCstains.pdf

This site is another good source containing TLCstain recipes.

Documents

Current Protocols in Nucleic Acid Chemistry || Thin-Layer Chromatography