hplc

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Kromatografi Cair Kinerja Tinggi (HPLC)

Extraction Distillation Precipitation Chromatography

Several different separation technique:

• HPLC adalah alat yang sangat bermanfaat

dalam analisis. Bagian ini menjelaskan

bagaimana pelaksanaan dan penggunaan

serta prinsip HPLC yang sama dengan

kromatografi lapis tipis dan kromatografi

kolom

Chromatography is an analytical chemistry techniques for the separation of mixtures. It involves passing the sample, a mixture which contains the analyte, in the "mobile phase", often in a stream of solvent, through the "stationary phase.“ Separation based on differences of migration of each component due to the difference of the nature (interaction) of each component to the stationary and, mobile phase

A,B,C

A B C

C

B

A

Mobile Phase

Stationary

phase

The stationary phase in HPLC

refers to the solid support

contained within the column

•the mobile phase continuously flows

CHROMATOGRAPHY: most widely used Qualitative and quantitative Many variety of compounds Good precision and accuracy Many choices of developing separation method

• HPLC merupakan perkembangan tingkat tinggi dari kromatografi kolom. Selain dari pelarut yang menetes melalui kolom di bawah grafitasi, didukung melalui tekanan tinggi sampai dengan 400 atm. Ini membuatnya lebih cepat. HPLC memungkinkan penggunaan partikel berukuran sangat kecil untuk material terpadatkan dalam kolom yang akan memberi luas permukaan yang lebih besar berinteraksi antara fase diam dan molekul-molekul yang melintasinya. Hal ini memungkinkan pemisahan yang lebih baik dari komponen-komponen dalam campuran. Perkembangan yang lebih luas melalui kromatografi kolom mempertimbangkan metode pendeteksian yang dapat digunakan. Metode-metode ini sangat otomatis dan sangat peka.

CLASSIFICATION

Mobile Phase Gas Liquid Samples Volatile - Less volatile Thermally stable - Less thermally - High molecular weight compound - ionic species Collection(rec.) Less convenient Easy Column Limited Greater variety Interaction One phase Two phases Operating temp. High room temperature Detector Limited Choice More Choices

Parameters GC LC

The differences between modern and classical LC

Classical LC Column used only once Solvent flow gravity/cappilarity Detection manually, coloring Quantitation Less precise, accuracy speed of Sep Slow Resolution low

HPLC reusable Pump Continuously More Faster Better

MODERN VS CLASSICAL LC

Bed Preparation

Sample application Solvent flow

Detection, quantitation

Result

Color Form

TLC

HPLC

MECHANISM OF SEPARATION IN HPLC

EXCLUTION CHROMATOGRAPHY

Separation are based on a sieving process If Solute is larger than the largest pores, it will be excluded and smaller molecule can enter a few pore have longer retention time.

Small M Large BM

THF (Molecular Sieving/GPC: Gel Permeation

Chromatography)

Adsorption Partition

Ion Exchange Exclution

High performance liquid chromatography (HPLC) Methods

High performance liquid chromatography (HPLC) Methods

Fase normal HPLC

sama dengan kromatografi lapis tipis atau

kromatografi kolom. Kolom diisi partikel silika yg

sangat kecil 7 pelarut non polar misalnya heksan.

Kolom in 4.6 mm panjang 150 sd. 250 mm.

Senyawa-senyawa polar dalam campuran melalui

kolom akan melekat lebih lama pada silika yang

polar dibanding dengan senyawa-senyawa non polar.

Oleh karena itu, senyawa yang non polar kemudian

akan lebih cepat melewati kolom.

Fase balik HPLC( bentuk yang biasa digunakan dalam HPLC)

ukuran kolom sama, tp silika (fase diam) dimodifikasi menjadi non

polar melalui pelekatan rantai-rantai hidrokarbon panjang pada

permukaannya secara sederhana baik berupa atom karbon 8 atau 18.

Sebagai contoh, pelarut polar digunakan berupa campuran air dan

alkohol seperti metanol.

Dalam kasus ini, akan terdapat atraksi kuat antara pelarut polar dan

molekul polar dalam campuran yang melalui kolom. Atraksi yang

terjadi tidak sekuat atraksi fase diam dan molekul-molekul polar

dalam larutan. Oleh karena itu, molekul-molekul polar dalam

campuran akan menghabiskan waktunya untuk bergerak bersama

dengan pelarut.

Fase balik HPLC( sambungan)

Senyawa-senyawa non polar dalam campuran akan cenderung

membentuk atraksi dengan gugus hidrokarbon karena adanya

dispersi gaya van der Waals. Senyawa-senyawa ini juga akan

kurang larut dalam pelarut karena membutuhkan pemutusan ikatan

hydrogen sebagaimana halnya senyawa-senyawa tersebut berada

dalam molekul-molekul air atau metanol misalnya. Oleh karenanya,

senyawa-senyawa ini akan menghabiskan waktu dalam larutan dan

akan bergerak lambat dalam kolom.

Ini berarti bahwa molekul-molekul polar akan bergerak lebih cepat

melalui kolom.

CHARACTERISTIC OF NORMAL AND REVERSE PHASE

STATIONARY PHASE MOBILE PHAS E TYPICAL STATIONARY

TYPICAL MOBILE PHASE

NORMAL PHASE polar

nonpolar

silica gel alumina

hydroxyapatite

Hexane Chloroform iso-octane

REVERSE PHASE

nonpolar

polar

-C-18 -C-8

-Phenyl -CN

methanol

water acetonitryl

O

Si-OH

Si-OH OH

Hexane

OH

C

CH3

CH3

CH3 C

CH3

CH3

H3C

CH3

NORMAL PHASE LIQUID SOLID

Sample with high polarity will be retained

Si-O-Si-C18

OH

CH3CN/H2O

OH

C

CH3

CH3

CH3 C

CH3

CH3

H3C

CH3

REVERSE PHASE

Sample with high polarity will be eluted easily

Reverse phase most widely used Polar, nonpolar, neutral, ionic compound

Ionic compound by reverse

phase Ion suppression Ion pairing Ion excgange

Ion pairing

X+ + Y- XY

Ionic Sample Counter Ion neutral

HA H+ + A- Neutal PH << Ionic

ION SUPPRESSION

CH3CN /H20

COO- COOH

Si-O-Si-C18 COOH

Lower pH

Benzoate ion Benzoic acid pH 2.5

Column : Novapak C18 Mobile Phase : CH3CN/H2O With PIC A pH 7.7

ION PAIRING C4 C4

+N OH

C4 C4

CH3

l

N

N

l

CH3

COO-

ION PAIR Benzoic Acid

N

H3C-N

O

O

Si-O-Si-C18

C4 C4

+N OH

C4 C4

Caffein

ION PAIR REAGENTS

PIC B FOR BASES

N+

H2PO4-

SO3-

SO3-

SO3-

SO3-

STRONG BASE STRONG ACIDS

PIC A FOR ACID

Pentan Sulfonat Acid

Hexane Sulfonic Acid

Heptane Sulfonic acid

Octane Sulfonic Acid Tetra Butyl Amonium

Phosphate

CHEMICAL STRUCTURE

ION EXCHANGE THEORY

SO3- , COO-

Cation Exchange

-(R)2NH+

Anion Exchange

X - + R+Y- Y- + R+X- (anion exchange)

X + + R-Y+ Y+ + R-X+ (Cation exchange)

D:/04-MY PRIVATE_DOC/01-AJAR/UNIT INSTRUMENTASI/Yohan/anis/uranium di jepang.ppt

Retention Time

Waktu yang dibutuhkan oleh senyawa untuk bergerak melalui kolom

menuju detektor disebut sebagai waktu retensi. Waktu retensi diukur

berdasarkan waktu pd saat sampel diinjeksikan sampai sampel

menunjukkan ketinggian puncak yang maksimum dari senyawa itu.

Senyawa-senyawa yang berbeda memiliki waktu retensi yang berbeda.

Untuk beberapa senyawa, waktu retensi akan sangat bervariasi dan

bergantung pada:

tekanan yang digunakan (karena itu akan berpengaruh pada laju alir dari

pelarut)

•kondisi dari fase diam (tidak hanya terbuat dari material apa, tetapi juga

pada ukuran partikel)

•komposisi yang tepat dari pelarut

temperatur pada kolom

Retention Time

the retention time (tR ),

the time taken for the

mobile phase to pass

through the column is

called tM.

capacity factor = k'A = t R - tM / tM

A term called the retention factor, k', is

often used to describe the migration rate of

an analyte on a column

Column Efficiency

Column efficiency, also known as plate count, is a measure of the

dispersion of a peak. Narrow peaks take up less space in the

chromatogram and thus allow more peaks to be separated. They are also

easier to integrate since they give better resolution and less overlapping.

Efficiency is usually explained using the concept of theoretical plates.

This model supposes that the column contains a large number of separate

layers. Separate equilibrations of the sample between the stationary and

mobile phase occur in these plates.

where t is the retention time of the peak of interest and W is the

peak width at the base

Theoretical plate

Similar to the measurement

for resolution, the

measurement for efficiency

may also be performed

using the peak width at half

height, where Wh/2 is the

peak width as half height

Resolution

Resolution

• Chromatogram = A plot of the detector’s signal as

function of elution time or volume.

• Retention time = The time a solute takes to move

from the point of injection to the detector (tr).

• Retention volume =The volume of mobile phase

needed to move a solute from its point of injection to

the detector (Vr).

• baseline width = The width of a solute’s

chromatographic band measured at the baseline (w).

In relation with the physical parameters

expressed as follow:

'

' N

41 R

Selectivity efficiency Capacity

OPTIMISING RESOLUTION

By MODIFYING κ’, α and N

A. κ1 (RETENTION FACTOR)

CHANGE SOLVENT RATIO / MIXTURES

(SOLVENT STRENGTHS) E.G. 50/50 MeOH/H20 - 70/30 MeOH/H20

B. α (SELECTIVITY FACTOR)

• CHANGE MOBILE PHASE CHEMISTRY

(CHANGE SOLVENT TYPES)

E.G. MeOH/H20 - THF/H20ADD

• BUFFER/MODIFIER

• CHANGE COLUMN TYPE

C. N (EFFICIENCY FACTOR):

• CHANGE FLOW RATE (DECREASING F.R. INCREASES

EFFICIENCY)

• CHANGE PARTICLE SIZE (DECREASE PARTICLE

SIZE INCREASES EFFICIENCY

• CHANGE TO A LONGER COLUMN (INCREASES

COLUMN LENGHTH INCREASE EFFICIENCY)

0

01

V

V - V ',tentionRe

01

02

V- V

V - V

'

' ',ySelectivit

2

1

1

W

V16 N,Efficiency

The physical meaning

κ1 = 0 (No separation, compound elutes at void vol. (vow))

κ1 = 2 - 10 (recommended value)

α = 1.0 (no separation)

α = 2.0 (good peak separation)

N = 3000 – 10.000 plates / column

R = 1.5 (baseline separation)

HPLC COMPONENT

EQUIPMENT AND OPERATION

Pumps

material :stainless steel, titanium or some ceramic

materials that are resistant to corrosive

Flow rate : 0.1 ml/min to 5-10 ml/min in analytical column

(i.d. 4-5 mm) deviation : less than 0.5-1%.

Reservoir : inert, mobile phase degassed , vacuum,

purge by helium or ultra sonic

Sample Injector

Chromatographic column

id are 2 to 3 mm lengths are 5-30 cm

packing material with diameter 3-10 m

Detector

Selective detector with signal proportional

to the concentration of only specific substance

in eluate

Universal (non specific) detector, with

response proportional to the a certain overall

property of the eluate, i.e. of both the solute

and the component of mobile phase. (RI)

UV/VIS detector Io

Log = bc = A

I

Io = incident light

I = the itensity of the

transmitted light

= Molar absortivity

b = Cell pathlength

c = Sample concentration

A = Absorbance

Fixed and Variable

Wavelength and diode

array

Data Processing Devices

Borwin sofware

Qualitative and quantitative Analysis

• tR is characteristic of the substance with direct

comparation to standard

• quantitative analysis the base on measurement of peak

heigh or peak areas value

1. TURN ON THE POWER

2. PREPARE FILTERED AND DEGASSED SOLVENT, AND

FILTERED SAMPLE

3.”PRIME” PUMP TO REMOVE BUBLE

4. ADJUST FLOW RATE COMPISITION OF SOLVENT

AND WAVELENGH USING BORWIN SOFTWARE

5. EQUILBRATE THE SYSTEM BY FOLLOWING THE

WORKING SOLVENT

6. INJECT STANDARD/ SAMPLE

7. INTEGRATE AND PRINT CHROTOGRAM RESULT

(y-RAM SOFTWARE)

OPERATON OF HPLC

SAMPLE PREPARATION

The purpose : to clean up the sample

Reduce overall sample load onto column

Remove interference associates with matrix

Concentrate the component of interest

Enhance sensitivity

Remove particulate

Protect the column

Improve the chromatographic separation peak

Type of sample clean up

Extraction

Filtration

TLC

Selective precipitation

Open column technique

Centrifugation

Guard column

A. Solvent

Have high purity, HPLC grade

Miscible each other if using more different solvent

pH 3,5 – 7,0 ( bonded phase)

UV adsorption

Toxicity

Volatility

A.1. H2O as a solvent

Use high quality of water (bidest)

Prepared freshly daily

Use appropriate containers

PRACTICAL HINT FOR AINTENANCE

A.2 Degassing

Purpose :

To removed gasses especially O2 and N2

To prevent bubble formation in pump and fluid system

To reach great precision of retention time and quantitation

Type: Helium Spurge, Vacuum, Sonication

A.3. pH Consideration

Reverse and normal phase 3,5 - 8.0 (8< pH

silica solubility ; PH < 2 bonded destroyed)

Ion exchange pH: 2 - 12

A.4 Solvent miscibility

A.5 Use of buffer : To improve Resolution

High quality, fresh, avoid precipitation

C. Column

C1. Column handling

To maximize the column lifetime

Avoid mechanical, thermal and pressure shocks

Always increase solvent flow rates slowly (0.1- 0.5 incr.)

Follow manufacture instruction

C2. Guard column

Protect column from particulate and chemical contaminant

Sample clean up

Trace enrichment using proper guard column

B. Filtration: remove particulate

To maintain life time column and pumping system

Type : Teflon, Nylon, cellulose

C3. Column cleaning

.Reverse phase column

Flush with 1005 Org. Solvent as MeOH for 15 min

Contaminated non proteinaceous material

Sequence cleaning ;

Wash solvent Volume

Water 30 ml

Methanol 30 ml

THF 30 ml

Methylene chloride 30 ml

THF 30 ml

Methanol 30 ml

Water 30 ml

Proteinaceus material : injecting 200 ul sludge of DMSO

Normal phase column

Sequence cleaning ;

Wash solvent Volume

Isooctane 50 ml


Methanol 50 ml


Isooctane 50ml

Solvent should be dry ( less than 500 ppm water)

Store the column in appropriate solvent ( Manual)

Store with end fitting connected

Protect from mechanical, thermal shock and

vibration

Flush column thoroughly prior to storage

Leaving column less 72 hours does not require

storage procedure

C4. Column storage recommendation

Istilah-istilah

• Chromatogram = A plot of the detector’s signal as

function of elution time or volume.

• Retention time = The time a solute takes to move

from the point of injection to the detector (tr).

• Retention volume =The volume of mobile phase

needed to move a solute from its point of injection

to the detector (Vr).

• baseline width = The width of a solute’s

chromatographic band measured at the baseline

(w).

Soal-soal

hplc

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