HPTLC PPT

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SEMINAR ON HPTLC

MAHARSHI ARVIND INSTUTE OF PHARMACY

MANSAROVAR, JAIPUR.

GUIDED BY

_.

Prepared by

Mr.

Kap

Kapil

il

sh

sharm

arma.

a.

Jas

Jasmi

min

n .H.

.H.

modi.

M pharma

(

Pharmaceutics)Pharmaceutics) I semester I semester

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Contents.

  

 Introduction.Introduction.Introduction.Introduction. 

 Introduction to H.P.T.L.C.Introduction to H.P.T.L.C. 

 

 Principle.Principle.Principle.Principle. 

 Selection of HPTLC plates.Selection of HPTLC plates. 

 Activation of pre coated plates.Activation of pre coated plates. 

 SAMPLE PREPARATION:SAMPLE PREPARATION: 

 Evaluation of spot or band.Evaluation of spot or band. 

 Application of HPTLC.Application of HPTLC. 

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Introduction:

chromatography

chromatography iiss a phya physsicical pr al pr ooccessess of of seseppeerarattiionon iinn

wh

whicichh tthhee ccomompponentonent iiss toto bbee seseparaparatedted ar ar ee ddiistusturbrbetedeted

b

betetwweeneen ttwwoo iimmmmiissciciblblee aa stationary phasestationary phase wwiitth hah hass larglargee

su

sur r f f aaccee ar ar eea aa andnd aa mobile phasemobile phase whwhicichh iiss iinn cconstonstaantnt

mot

motiionon tthr hr ououghgh tthhee ststaattiiononary phaary phase.se.

Introd

Introduction uction to to H.PH.P.T.T.L.C .L.C H.P.T.L.C

H.P.T.L.C _i_i_s_s _t_t_h_h_e_e _i_i_m_m_pr_pr_oved_oved _met_met_h_h_od_od _of_ofT.L.CT.L.C _wh_wh_ic_ic_h_h _ut_ut_i_i_l_l_iz_iz_e_e _t_t_h_h_e_e

c

conventonventiiononalal tetecchhnniqiqueue of of TT CC iinn momor r ee ooppttiimmizizee wayway ..

I

Itt iiss alalsoso kknonowwnn aass a plaa planener r cchr hr omomaatotographygraphy oor Flar Flat-t- babadd

c

(5)

´

Principle

HP

HP_T_T _C_C _t_t_ak_ak_e_e _pla_pla_c_c_e_e _i_i_n_n _h_h_i_i_gh_gh _s_s_p_p_eed_eed _c_c_ap_ap_i_i_llary_llary _f_f_l_l_o_o_{w ra}_{w ra}_n_n_g_g _of_of_t_t_h_h_e_e

mo

mobbiillee phaphasese

,

ThTheer r ee ar ar ee tthr hr eeee mmaaiinn stestepp iinn HPHP_T_T _C_C

1]

1] ssaammplplee toto aannalyalyzzeded toto cchr hr omomaatotogragramm laylayeer r vovollumeume

pr

pr eecicissiionon aandnd susuiittablablee ppososiittiionon ar ar ee aacchhiievedeved byby useuse of of

su

suiittablablee iinstnstr r ument.ument.

2]so

2]sollventvent ((momobbiillee phaphase)se) mmiigragratestes tthhee plaplannednned ddiiststaannccee iinn

lay

layeer(r(ststaattiiononaryary)) byby ccapapiillary allary accttiionon iinn tthhiiss pr pr ooccessess ssaammplplee

se

separaparatedted iinn iitsts ccomompponents.onents.

3]

3] seseparaparattiionon ttraracckkss ar ar ee ssccaannednned iinn densdensiitometetometer r wwiitth lh liighghtt

b

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S

_{election of}

HPTLCHPTLC

_plates

..

Previously ha

nd made plate is

used in TLC

for

both qualitative and quantitative work, certain

draw back with that is non uniformly layer ,

form

atio

n of th

ick lay

er pav

ed for a

dvan

t

pre

coated plates.

Now a days pre

coated plates are available in

dif

feren

t fo

rmet

and th

ickn

ess by dif

feren

t

manufa

ctures. these plates are

used for both

qualitative and quantitative purpose in

HPTLC.



 _g_g

lass plates .



Polyester /polyethylene.



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G

GLALASSSS PLATEPLATESS



 Resistance to heatResistance to heat



 Easy Easy to to handle handle Thickness Thickness 1.3mm1.3mm



 Offer superior plane and smooth Offer superior plane and smooth surface.surface.

Fra

Fraggileile

Hi

Higgh weih weigghtht

Hi

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´

´ POLYPOLY

T

POLYEPOLYE

T

HYLENEHYLENE

.

Thickness of plate 0.2mm

It can be produce in Roll form.

Unbreakable.

Less packin

gg

material required.

Development of plate is not above temp. 120

Development of plate is not above temp. 12000losses oflosses of

its shape

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´

´ A A

l

um

i

n

i

um

pla

tes.

Thickness of plate 0.1mm

It can be produce in Roll form.

Unbreakable.

Less packin

Less packingg material required.material required.

Development of plate is not above temp. 120

Development of plate is not above temp. 12000losses of itslosses of its

shape.

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S

_o

_rb

_ents

_used

_i

_n

HPHP

_T

LL

_C

PP

_la

_tes.

Sorbent used in conventional

TLC can be used in HPTLC with or

with out modification.



 SilicaSilica ggel 65F(modified)el 65F(modified)



 HiHigghly purified silicahly purified silica ggel60.el60.



 Aluminium oxide.Aluminium oxide.



 Microcrystalline.Microcrystalline.



 SilicaSilica ggel Gel G particle sizeparticle size of sorbent

of sorbent



 Reversed stationary phase.Reversed stationary phase. HPTLC HPTLC 6 6 mm



 Hybrid platesHybrid plates. . TLC TLC 10 10 mm

Layer thickness in HPTLC 100200 m

in TLC 250 m

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´

´ LL

ay

e

r pr

e

wa

s

h

i

g

.



 AscendinAscendingg method .method .



 continuous method.continuous method. 

 DeepinDeepingg method.method.

solvent used for washin

solvent used for washingg

methanol methanol chloroform: methanol:amonia(90:10:1) chloroform: methanol:amonia(90:10:1) Chloroform: methanol(1:1) Chloroform: methanol(1:1) Ammonia solution (1%) Ammonia solution (1%)

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A

_c

_t

_i

_v

_a

_t

_i

_on

_of

_pr

_e

_c

_o

_a

_ted

_pla

_tes

_.

The plates are activated by placin

The plates are activated by placingg in oven at 110in oven at 110

120

12000c for 30 minutes,this step will remove the waterc for 30 minutes,this step will remove the water

that has been physical absorbed on the surface at

solvent layer.

Freshly open box of HPTLC plates usually not requird

activation.

Activation at hi

Activation at higgher temperature and lonher temperature and longg time istime is

avoided which may tends to vary active layer and

sample decomposition .

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SAM

SAMPLEPLE PREPPREP A ARR A A

_TI

ON:ON:

Proper sample preparation is pre requsite for the

success HPTLC separation.

Beside maximizin

Beside maximizingg the the yield yield of of analyte analyte in in thethe

selected solvent ,stability of the analyte durin

selected solvent ,stability of the analyte duringg

extraction and analysis must consider. there for

choice

choice of of suitable suitable solvent solvent forfor ggiven analysis is veryiven analysis is very

important .

Solvent for dissolvin

Solvent for dissolvingg the sample should be nonthe sample should be non

polar and non volatile as far

polar and non volatile as far as possible since polaras possible since polar

solvent are likely to induce circular chromato

solvent are likely to induce circular chromatoggramram

at the ori

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A

_ppl

_ic

_a

_t

_i

_on

_of

_s

_a

_m

_pl

_e

_a

_nd

_st

_a

_nd

_ar

_d

_so

_l

_ut

_i

_on.

Sample application is one imp and critical step for

obtainin

obtainingg thethe ggood ood resolution resolution for for quantification quantification byby

HP

HPTLTLC. saC. sampmple/le/stdstd arare ape appliplied aed as sps sporort or bat or bandnd

dependin

dependingg upon the analysis spot application is doneupon the analysis spot application is done

by usin

by usingg

1)

1) Capillary tubes.Capillary tubes. 2)

2) Micro bulb pipetts.Micro bulb pipetts. 3)

3) Micro syrinMicro syringg..

4)

4) Automatic sample applicator.Automatic sample applicator.

com

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C

AMA AMANGNG LL

_I

NONOMAMA

_T

Caman

Camangg inomat inomat with with a a spray spray tech tech isis

Automated sample application device. Automated sample application device. The sample is loaded in micro syrin The sample is loaded in micro syringg

(Hamilton syrin

(Hamilton syringg )of 1.0 capacity.)of 1.0 capacity.

The sample is applied as a spot or band The sample is applied as a spot or band By pro

By proggramminrammingg instrument parameterinstrument parameter

Like spottin

Like spottingg volume ,band lenvolume ,band lenggth,th,

No of spot/band , space between spot/ No of spot/band , space between spot/ band.

band. FiFigg

CAMANG LINOMAT

CAMANG LINOMAT APPLICATOAPPLICATORR

The

The nozzle nozzle is is placed placed at at tip tip of of syrinsyringg ,air is comin,air is comingg

out at hi

out at higgh pressure atomize the sample solution inh pressure atomize the sample solution in

to fine spray to fine spray

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´

C

HROHROMAMA

T

OGROGR AM AM

D

EVELOPEVELOPMMENEN

T

.

After application of sample in HPTLC plate,

chromato

chromatoggram is developed by dippinram is developed by dippingg in suitablein suitable

solvent system

Taken in developin

Taken in developingg chamber.chamber.

The solvent system rises over the layer

The solvent system rises over the layer byby

capillary action and separation of

sample in different components

take place.

selection of solvent system

Chamber saturation

Type of development and developin

Type of development and developingg devicedevice..

F

Fiigg

:

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´

´ LL

I

NENE A ARR A ANN

D

RR A A

DI

A ALL

D

EVELOPEVELOPMMENEN

T

.

´

In close bad tech like HPLC linear

development is possible but an open bad

technique does not suffer this

limitation.

HPTLC can develop by

Ascendin

gg

.

Descendin

gg

.

Circular.

Anti circular

.

FI FI :: HHPPTTL L f f iinn nn ..

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´

D

ete

c

t

i

on

o

r

v

i

su

la

t

i

on of

s

p

ot/

ba

nd.

Ther

There is e is no dino diffuculffucultt in din detecetectintingg the colouredthe coloured

subst

substance.ance.oror colour lcolour les subses substance atance absorbsorbinbingg the uvthe uv

rad

radiatiiationoronor with fluowith fluoresceresce(Rib(Riboflaoflavin)vin)

Detection of spots/band are done by

Detection of spots/band are done by 1)

1) destruction/Non reverse.destruction/Non reverse.

2)

2) Nondestructive/reversible.Nondestructive/reversible.

3)

3) Misc. method.Misc. method.

FIG:HPTLC VISULIZER

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´

´ EE

v

al

u

a

t

i

on

of

s

p

ot

o

r ba

nd.

After detection of spot /band upon objective of

experiment chromato

experiment chromatoggram is used for severalram is used for several

purpose. purpose. Quality Evaluation . Quality Evaluation . Quantitative Evaluation . Quantitative Evaluation .

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´

´ A A

ppl

ic

a

t

i

on

of

HPHP

T

LL

C.

Pharmaceutical research. Pharmaceutical research. Biomedical Analysis. Biomedical Analysis. Clinical Analysis. Clinical Analysis. Environment Analysis. Environment Analysis. Food industry. Food industry. Therapeutic dru

Therapeutic drugg monitorinmonitoringg to determine itsto determine its

concentration and metabolites in blood urine

concentration and metabolites in blood urine etc.etc.

Analysis

Analysis of of environment environment pollution pollution level.level.

Quantitative determination of prosta

Quantitative determination of prostagglandin s andlandin s and

thr

thrombomboxaoxanesnes in plin plasmasma.a.

Determination of mercury in water.

Characterization of hazard in industrial waste.

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PARAMETER

PARAMETER TTLLC C HHPPTTLLCC

T

TYPEYPESS OO_F_F

C

CHROHROMAMA_T_TOGROGR A APHPH_IIC_C PL PL A A_T_TEESS H H A ANN_D_DMAMA_D_DEE_//PP RE RE_C_COO A A_T_TEE_D_D PRE

PRE_C_COO A A_T_TEE_D_D

ABS

ABSORORBBENEN_T_T LL A AYERYER _200-250_{200-250 m}_m ₁₁₀₀₀_0--1₁₅₅₀_0m_m P

P A ARR_TI_TIC_CLELE SS_IZ_IZEE RR A ANGENGE _5-20_{5-20 m}_m ₄_4--8_{8 m}_m A

APPLPPL_IIC_C A A_TI_TIONON OO_F_F SAM

SAMPLEPLE

MA

MANUNU A ALL_//SSEEMM_II A

AUU_T_TOOMAMA_TI_TIC_C

MA

MANUNU A ALL_//SSEEMM_II A AUU_T_TOO MA

MA_TI_TIC_C S

SHH A APEPE OO_F_F SAMSAMPLEPLE SSPOPO_T_T SSPOPO_T_T//BABANN_D_D S

SPOPO_T_T SS_IZ_IZEE ₃_3--6_6m_mm_m ₁_1--2_2m_mm_m SAM

SAMPLEPLE VOLUVOLUMMEE ₁_1--1₁₀₀ ₀_0..1_1--2₂

DIFF

ERENEREN

_C

EE BBEE

_TW

EENEEN

_T

LL

_C

A ANN

_D

HP

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PARAMETER

PARAMETER TTLLC C HHPPTTLLCC

NO

NO OO_F_F SAMSAMPLEPLE OEROER PLPL A A_T_TEE ₁₁₅_5--2₂₀₀ ₄₄₀_0--4₄₅₅ OP

OP_TI_TIMAMALL _D_DEVELOPEVELOPMMENEN_T_T

DI

DISS_T_T A ANN_C_CEE

10-15

10-15ccmm. . 55--77ccmm

D

DEVELOPEVELOPMMENEN_{T TI}_{T TI}MMEE _D_DEPENEPEN_D_D UPONUPON M

MOOBB_IILE PHLE PH AS ASEE

40

40 %% LELESSSS _T_TH AH ANN _T_TLL_C_C

Q

QUU A ANN_TIT_TIT A A_TI_TIONON MAMANUNU A ALL MAMANUNU A ALL

//IINNSS_T_TRURUMMENEN_TI_TIONON_.. REPRO

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´

´ RR

efe

r

en

c

es.

´

´ PrinciplePrinciple of of IInstnstr r umentumental aal annalyalytticicalal ,s,skkoooogg,, HHoolllleer r ,,

N

N_i_i_em_em_a_a_n._n.

´

´ IInstnstr r umentumentalal metmethhodod of of aannalyalyssiis,s, wwiillar llar d,d, memerr rr iietet ,de,deaan.n.

´

´ PPhar har mmaacceuteuticical aal annalyalyssiis,munson.s,munson.

´

´ IInstnstr r umentumentalal metmethhodod of of cchhememicical aal annalyalyssiiss gguur r udeeudeepp

c

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