HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High-pressure liquid chromatography).
High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.
It is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a mixture.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High-pressure liquid chromatography).
High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.
It is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a mixture.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
Introduction to High Performance Liquid Chromatography-HPLCRoyan Institute
This presentation is a simple explain of HPLC which introduce this method easily. You can use this PPTx File to present in your class and seminars as well. I prepare this file to present in Tabriz University of Medical Sciences when I was a MSc Medical Nanotechnology student. It will be useful for you too.
This presentation lays out all the differences between the two analytical techniques i.e. High Performance Thin Layer Chromatography and High Performance Liquid Chromatography.
For more information regarding HPTLC, please visit www.anchrom.in or you can send us an email at lab@anchrom.in
Thank you
Anchrom
Usually, analysis is not considered an easy subject and it can't be understood on its own if you don't have some proper notes and clear concepts so I am here to help you in analysis for clearing few concepts on UV-Visible spectrophotometer, soon will come up with a new set of notes on new topic depending upon the response.
Introduction to High Performance Liquid Chromatography-HPLCRoyan Institute
This presentation is a simple explain of HPLC which introduce this method easily. You can use this PPTx File to present in your class and seminars as well. I prepare this file to present in Tabriz University of Medical Sciences when I was a MSc Medical Nanotechnology student. It will be useful for you too.
This presentation lays out all the differences between the two analytical techniques i.e. High Performance Thin Layer Chromatography and High Performance Liquid Chromatography.
For more information regarding HPTLC, please visit www.anchrom.in or you can send us an email at lab@anchrom.in
Thank you
Anchrom
Usually, analysis is not considered an easy subject and it can't be understood on its own if you don't have some proper notes and clear concepts so I am here to help you in analysis for clearing few concepts on UV-Visible spectrophotometer, soon will come up with a new set of notes on new topic depending upon the response.
HPLC Instrumentation, Pharmaceutical analysis, HPLC detectorsInstmn sem sharu(4)
Coloane utilizate in HPLC.pptx
1. Coloane si sisteme de injectie utilizate in HPLC
Prezentul material este folosit în scop didactic și se adresează în numai studentilor inscriși la acest curs.
2. Instrumentatie
Componentele HPLC
Probele lichide sunt injectate in coloana cu o siringa printr-o valva de injectie. Proba este purtata de-a
lugul coloanei cromatografice de faza mobila, iar separarea cromatografica are loc pe masura ce
amestecul traverseaza coloana. Detectorii in HPLC detecteaza componentul eluat la capatul coloanei pe
baza unor proprietati fizice ca: absorbtia de lumina UV, fluorescenta, sau diferentele de indice de refractie
intre analit si faza mobila.
Schema unui sistem HPLC este prezentata mai jos:
Faza mobila
Rezervor
Pompa Injector Pre-Coloana Coloana Detector
Achizitie Date
5. Rezervoare de faza mobila, filtrare
Cele mai utilizate rezervoare de faze mobile sunt din sticla cu dopuri speciale, tuburi de teflon si filtre
care permit conectarea la pompa si purjarea gazului inert (heliu) pentru eliminarea aerului dizolvat.
Pulverizarea este un alt proces prin care gazele dizolvate sunt inlaturate din solvent prin barbotarea
unui gaz inert. Purjarea de He si stocarea solventului sub He nu este suficienta pentru degazarea
solventilor aposi. Sunt tinuti sub vid 5-10 min si apoi pastrati in atmosfera de He.
6. Sistem de furnizare solventi
Pompa JASCO LC-1580 Pump si detector UV-1570/1575 UV/Vis
http://www.jasco.co.jp/English/main/main.html
http://www.jasco.co.uk/lcpumps.htm
8. Pompe utilizate in HPLC
Printre pompele utilizate in HPLC se pot enumera:
Pompe cu piston,
Pompe tip siringa,
Pompe la presiune constanta
9. Pompele moderne prezinta parametrii:
Domeniul de debite: 0.01 - 10 ml/min
Stabilitatea debitului: nu mai mult de 1%
Presiunea maxima: < 5000 psi
Este de dorit un sistem de degazare integrat, cu purjare de He sau degazare la vid.
10. High-pressure piston pump for HPLC.
Solvent at the left passes through an electronic valve synchronized with the large piston and designed to minimize the formation of solvent vapor bubbles
during the intake stroke. The spring-loaded outlet valve maintains a constant outlet pressure, and the damper further reduces pressure surge. Pressure surge
from the first piston are decreased in the damper that “breathes” against a constant outside pressure, Pressure surge are typically <1% of the operating
pressure. As the large piston draws in liquid, the small piston propels liquid to the column. During the return stroke of the small piston, the large piston
delivers solvent into the expanding chamber of the small piston. Part of the solvent fills the chamber, while the remainder flows to the column. Delivery
rate is controlled by the stroke volumes. [courtesy Hewlett-Packard Co., Palo Alto, CA.]
11. Bucla de injectare probe
Un injector de probe manual tipic este compus dintr-o valva cu doua pozitii cu bucle de
injectare de 500 µl. Intr-o configuratie, faza mobila de la pompa este trimisa direct in coloana,
utilizandu-se valve cu actionare electrica sau pneumatica.
Injectorii automati de probe (autosampler) pot stoca si injecta secvential probe multiple, fiind
utilizati in controlul calitatii produselor.
13. Exemple de injectori in HPLC de tip Rheodyne: injector manual si injector cu bucla de injectie
Scale Sample Volume
Micro 2 µL to 500 µL
Analitic 1.0 µL to 5.0 mL
Preparativ 100 µL to 10 mL
http://www.rheodyne.com/sampinj.html
14. Diferite tipuri de varfuri de ace pentru siringile Hamilton utilizate in
functie de natura probelor de analizat.
http://www.hamiltoncomp.com/product/syringe/point.html
15. Bucla de injectie in HPLC
Reprezinta principala cauza a erorilor de volum de injectat in coloana cromatografica.
Proba se introduce in bucla, aceasta fiind inchisa pentru fluxul de faza mobila din HPLC.
Dupa ce bucla este umpluta, ea este re-deschisa pentru fluxul de faza mobila, proba fiind astfel introdusa in
coloana cromatografica din instrumentul HPLC.
PEEK™ Loop
Note:PEEK™ (polyetheretherketone) is a
registered trade mark of Victrex plc.
16. COLOANE HPLC
In HPLC, separarea se realizeaza pe baza diferentei de afinitate relativa a componentilor
probei fata de faza mobila sau fata de faza stationara. Mecanismele de separare au fost
prezentate anterior.
• Pentru aplicatii generale in faza normala si in faza inversa,
• Pentru toate aplicatiile de schimb ionic,
• Pentru excludere sterica si permeatie in gel,
• faze stationare semi-preparative si preparative sunt de asemeni disponibile.
17. Coloane in HPLC
Sunt si alte tipuri de coloane decat cele de separare propriu-zise care contin faza stationara: de
protectie, de derivatizare, capilare, rapide, si preparative.
Coloanele de protectie sunt plasate inaintea coloanei de separare fiind utilizate la protejarea
acesteia in scopul filtrarii sau separarii: 1) particulelor care pot infunda coloana; 2) compusilor care
conduc la variatii ale liniei de baza "baseline drift", la rezolutii si sensibilitati scazute si la aparitia
picurilor false; 3) compusilor care pot provoca precipitarea la contactul cu faza stationara sau faza
mobila; 4) compusilor care pot fi co-eluati si produc picuri suplimentare interferand in detectie si in
analiza cantitativa. Aceste coloane trebuie schimbate regulat pentru a-si pastra calitatile de protectie.
18. Coloane de derivatizare - Derivatizarea pre- sau post- coloana reprezinta un aspect important al analizei
cromatografice. Reducand sau alterand compusul initial la molecule chimice inrudite, la fragmente
moleculare se pot obtine date complementare datelor analizei. In putine cazuri, etapa de derivatizatizare
poate conduce la date nesigure sau neconcludente, acest aspect conferind avantaje HPLC in comparatie
cu GC. Deoarece GC necesita analiti volatili, termic stabili sau analiti nepolari, derivatizarea este
necesara si in acele probe care nu au aceste proprietati. Acetilarea, sililarea, sau hidroliza acida reprezinta
exemple de tehnici de derivatizare.
Coloane capilare - Progresele in HPLC au condus la coloane analitice de mici dimensiuni, cunoscute ca
micro-coloane, coloanele capilare au diametre sub-milimetrice si sunt de trei tipuri: tubulare deschise, cu
umplere partiala si umplute. Acestea permit lucrul cu probe de ordinul nanolitrilor, lucrul cu debite mici
si volume mici de solvent. In aceste conditii insa toata instrumentatia trebuie miniaturizata debitele sunt
dificil de reprodus, lucrul cu gradienti de elutie nu este eficient si injectarea probelor trebuie facuta cu
grija.
19. Microcoloanele cu umplutura sunt utilizate pentru volume mici, diametrul lor fiind de 1-2 mm. Ca
si coloanele capilare, instrumentele trebuie adaptate la folosirea unor coloane de acest tip (de ex.
debite mici in coloana). Pe langa avantajele volumelor mici de proba si de faza mobila se remarca o
crestere sensibilitatii fara a se pierde din rezolutie.
Coloane rapide - Argumentul in utilizarea acestor coloane este obtinerea unor probe superioare
(cantitate de component in unitate de timp). Pentru multe coloane, cresterea debitului sau vitezei de
migrare prin coloana afecteaza negativ rezolutia si separarea in general. Din acest motiv, coloanele
rapide sunt utilizate in scopul reducerii timpului de analiza fara deviatii semnificative ale rezultatelor.
Aceste coloane au acelasi diametru intern, dar lungimi mult mai mici fiind umplute cu particule cu
diametrul de aprox. 3 µm. Avantajele includ sensibilitate marita, timpi de analiza redusi, utilizarea
unor volume mai mici de faza mobila si reproductibilitate marita.
20. Coloane preparative - Aceste coloane sunt utilizate pentru probe de ordinul miligramelor in scopuri
preparative. O coloana preparativa are diametre mari pentru a facilita volume de injectie mari in
sistemul HPLC.
Accesoriile importante sunt regulatorul de presiune si colectorul de fractii. Primul este plasat imediat
dupa detector, in scopul asigurarii unei presiuni constante la iesirea din detector pentru a preveni
introducerea bulelor de aer in sistem si a imbunatati stabilitatea liniei de baza. Colectorul de fractii
colecteaza uniform fractiuni la iesirea din coloana, fiolele fiind plasate intr-un carusel programat in ceea
ce priveste intervalele de colectare. Fiecare fiola contine faza mobila si fractiunea din proba la timpul de
elutie corespunzator. Umplutura coloanelor are diferite dimensiuni de particule, diametre, dimensiuni
de pori sau suprafete modificate(in cromatografia de afinitate). (Brown, 1990).
21. Coloane
a. Coloanele sunt confectionate din otel inoxidabil.
b. Lungimile sunt intre 10 - 30 cm.
c. Diametrele interioare sunt intre
4 - 40 mm; pentru noile coloane de inalta performanta ele
sunt cuprinse intre 1 - 4.6 mm.
d. Dimensiunea particulelor este cuprinsa intre 5 - 10
micrometri.
HPLC column with replaceable guard column to collect irreversibly adsorbed impurities. Titanium frits distribute the liquid evenly over
the diameter of the column.
23. Tehnici de impachetare
• La presiune atmosferica
• In camp electric.
• Vibratii aplicate pentru omogenizarea umpluturii.
24. Coloane capilare utilizate in HPLC
E
A
B C
D D
A Material polymeric polietilenimina
B Coloana capilara din cuart
C Packing Material
D Frita
E Material transparent
25. Daca k‘ pentru component descreste, compusii
incep sa migreze succesiv prin faza stationara.
In comparatie cu elutia izocratica, rezolutia si
separarea sunt imbunatatite.
Elutia cu gradient si cea izocratica
• Elutia izocratica
– un singur solvent (sau amestec cu
compozitie constanta)
• Elutia cu gradient
– Modificarea continua a compozitiei
amestecului de solventi pentru a creste
taria elutiei.
26. Elutia izocratica intr-o separare prin HPLC a unui amestec de compusi organici la un debit de 1.0mL/min intr-o
coloana cu 0.46x23cm Hypersil ODS(C18 pe 5-m silica) la temperatura(~22℃)
27. Separarea in HPLC prin elutie izocratica a unui amestec de compusi organici la un debit de
1.0mL/min pe o coloana de 0.46x23cm Hypersil ODS(C18) la temperatura(~22℃)
28. Separarea in HPLC prin elutie izocratica a unui amestec de compusi organici la un debit de 1.0mL/min pe o coloana de
0.46x23cm Hypersil ODS(C18 on 5-m silice) la temperatura(~22℃)
29. 8 13 28 30
Pe masura ce creste concentratia CH3CN, scade si solubilitatea componentilor de separat in faza
mobila si creste retentia acestora pe faza stationara.
30. Cromatografie de schimb ionic de inalta performanta
1. Coloana contine o faza stationara de tip
a. rasina polistirenica grefata cu grupari schimbatoare de ioni, granulata,
cu dimensiuni de granule <10 micrometri.
32. 2. Probele sunt trecute prin coloana ce contine ca faza stationara schimbatori de ioni, aparand urmatoarele reactii de
schimb ionic:
a. In the top diagram above, the resin would be exhaustively washed with acid to "load" the cation exchange sites
(sulfonic acid groups in the example above) with protons (and elute any bound cations).
b. In the lower diagram, a sodium-containing sample is then loaded on the column. The sodium competes and displaces
the protons and binds to the resin. The protons are then eluted.
c. The amount of sodium bound can be determined by titrated the eluted acid.
d. The law of mass action applies in all cases: high concentrations of one species shift equilibrium, favoring binding of
the higher concentration species and elution of the other.
33. 3. Ion-exchange is also commonly performed in open columns (without the pumps and automated controls
of the HPLC).
a. Polystyrene-type resins may be used, such as...
1) DOWEX 50 (a strong, cation exchanger with a sulfonic acid functional group).
2) IRC-150 (a weak, cation exchanger with a carboxylic acid functional group).
3) DOWEX 1 (a strong, anion exchanger with a quaternary amine functional
group).
4) IR-45 (a weak anion exchanger with a methylammonium functional group).
b. Polysaccharide-type resins may be used, such as...
1) DEAE-cellulose and DEAE-Sephadex (weak anion exchangers with the
diethylaminoethyl functional group). Sephadex is a trade name of a beaded,
cross-linked dextran bead.
2) CM-cellulose and CM-Sephadex (weak cation exchangers with the
carboxymethyl functional group).
3) Phospho-cellulose and phospho-Sephadex (strong cation exchangers with a
phosphoryl group).
c. Polysaccharide-type resins are commonly used in protein applications with open columns, but can also be
used in HPLC work. However, polysaccharides cannot stand up to high pressures. (The HPLC must be run
at pressures less than ~150 pounds per square inch).
34. Cromatografie de excludere sterica
A. Tehnica cromatografica mai noua.
B. Se poate clasifica in cromatografie de permeatie in gel, filtrare in gel,
cromatografie de excludere moleculara, cromatografie de excludere sterica.
C. In aceasta tehnica, coloanele sunt umplute cu particule poroase cu diferite
diametre ale porilor. Particulele sunt confectionate din:
1. Polistiren - divinilbenzen (semirigid) rezistand la presiuni mari.
a. Solventii organici utilizati sunt: acetona, tetrahidrofuran, etc.), nu apa care nu
gonfleaza polistirenul.
b. Se lucreaza cu debite mici.
2. Sticla sau silice
a. Silicea permite debite si presiuni mari.
b. pH –ul trebuie mentinut sub ~7.5 peste care silicea se dizolva.
35. 3. Gelurile moi(dextrani, polizaharide sau polimeri similari).
a. Acestea includ:
1) Sephadex (dextran)
2) Sepharose (agarose)
3) BioGel P (polyacrylamida)
b. Presiunile se mentin scazute pentru a preveni aglomerarea gelurilor.
c. Sunt utilizate pentru specii solubile in apa cu mase moleculare cuprinse intre
100 - 25,000,000 grams/mol(e.g., proteine, enzime, etc.)
36. Cromatografie de lichide cu formare de perechi ionice
Q+ + X- = Q Xaq = QXorg
(faza mobila) (faza stationara)
k' = ([QX]org / [X]aq) (VS / VM)
= (VS / VM) E [Q+]aq
Counter ion ( cca 0.1 w/w % )
Probe acide ce contin cationi: saruri de tetraetil amoniu,
fosfat de tetrabutil amoniu
Probe bazice ce contin anioni: octil sulfonat, acid percloric
37. Separari cromatografice de amestecuri de compusi ionici si nenionici prin cromatografie cu perechi de ioni
38. Referinte bibliografice:
"Quantitative Chemical Analysis", D.C. Harris, Freeman, 9th Edition, 2015.
" Practical problem solving in HPLC" , S. Kromidas, WILEY-VCH, 2005
« Analiza instrumentala - Aplicatii», Ion Ion, Alina Catrinel Ion, Dragos Stefan
Nicolae, Editura Printech, Bucuresti, 2007
« Metode de separare si analiza cromatografica », Victor David, Andrei Medvedovici
, Editura Universitatii din Bucuresti, 2007