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High Performance Liquid

ChromatographyBarba, Biadomang, Dulos

CHEM 127.1

Monday, September 12, 2011

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Caffeine

belongs to a group of

alkaloids called xanthines

sample in this experiment

beverage


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HPLC

Before: large columns, large particles, under gravity feed,

manual collection of fractions of eluents

Giddings Prediction: smaller particles, under increased flow

pressure=high plate numbers


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HPLC

Csaba Horvath and

colleague S.R. Lipsky

built first HPLC

But, in 1970s

small silanized particlesallowed use of small-

volume columns=high

resolution


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Principles

In LC, rate of solute distribution between S.P and M.P

diffusion-controlledto minimize diffusion and time required for the movement of

sample and from interaction sites in the column

smaller eddy diffusion (small A value)

more rapid mass transport between the phases, i.e. high flow

rates (small C value)


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HPLC

B is usually small for liquids

small molecular diffusion

A is usually small and almost

constant for liquids


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Stationary phase

HPLC microparticles: high-purity silica

low in trace metal content

5 to 10 micrometers in diameter

pore sizes: 60 to 100 angstroms, 300 for large biomolecules

It can be partition (liquid-liquid) or adsorption

liquid S.P: coated on particles or chemically bound


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Stationary phase

microporous

10 micrometers

permeable to solvent

e.g. silica gel

perfusion

12 micrometers

larger molecules

nonporous

1.5 to 2.5 micrometers

for complex peptides

ion exchange

chromatography

monolithic columns

polymers

zirconia


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Components

Pump

forces the mobile phase

specific flow rate

isochratic or gradient

Injector

introduces liquid sample

autosampler

Column

Stationary phase

Detector

detect eluted

componentschromatogram

Computer


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Pump

Isocratic

preparative

Gradient

analysis


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Pump


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Types of HPLC

Reversed phase

nonpolar S.P. polar M.P.

S.P: aromatic or heterocycles

to provide pi to pi interactions

dispersive forces

Normal phase

polar S.P. nonpolar M.P.

polar forces

Ion exchange

based on charge and size

ionic forces

Size exclusion

based on size


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Detection in HPLC

Spectroscopic

UV absorption

MS detection

Refractive Index

RI: measure of moleculesability to deflect light

amount of deflection is

proportional to

concentrationFluorescence

only substance that

fluoresce


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Detection in HPLC


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HPLC


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HPLC can be used for quantitative and qualitative analysis

preparation of pure compounds

trace analysis


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HPLC

Examples of HPLC instrumentsMonday, September 12, 2011

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HPLC demonstration


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Methodology


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Preparation of Caffeine Standard

!"#$%&'(#()*$+")+()*#,-").$"/$+,'(&)($."01-").$

2$34456$7"0$8,.9.:$;(&:2?$

2:4?$@:2$,)%$34:4$5

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!"#$%&$'()*'*+,+-,./0'

$%&$'1.2'/(,+#345&67&8)0'

*+,+-,./'

9+)98:;8,?@AB'

'

!"#/+-./*+/0'9C.2'9$++*'/(,+D'

$/8./'8)E+-:.)0'(CC.2'FG',.'

$(99'HIJK=&8)9L0'

98&%C,()+.%9C

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Determination of Caffeine in Coffee and Tea

!"#$%&"'#()%*+'),'-#."/*"'/*'-).""'

'

012(3'-).""'/*'42(3'5)6'

7#$8'

'

'012(3'+"#'/*'42(3'5)6'7#$8'

'

!"#$%&"'#()%*+'),'-#."/*"'/*'+"#'

'

9/6%+"'+)'(#&8':;'

("+

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Determination of Caffeine in Cola Beverage

!"#$"%&'&&()*+%,-./%01(2%&"3"4#+"%)*%&"#5"46%71'4%%#*2%8149:%

#(9"4*#;3"

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!"##"$%&'()&%*+%,-%

%

%

,.(/%(0)(/*1(+'2%$3&4%

5"#61+%$7%891:%&"#;(+'%

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Results and Discussion


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Chromatogram


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Peak Area

Area under each single

response

Can be correlated to the

concentration of the sample


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Retention time

Distance of the peak maxima

from the injection point

expressed in time units

Serve as identifier for given

analyte on that particular

system

Most easily measurable

parameter

Retention volume (Vr)

!"#


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Red time/Void time (t0)

Retention of non-retained analyte

Part of the total analyte retention time that the analyte spends in

the mobile phase moving through the column

Serves as correction factor and allows validation of flow rate

i.e. thiourea, uracil, and NaNO3


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Retention Factor (k)

Or capacity factor

Dimensionless, and independent on mobile phase flow rate and

column dimensions

For reproducibility, characteristic of a chromatographic system


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Retention Factor (k)

Ratio of reduced retention time to void time


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Selectivity factor ()

Ability of chromatographic system to discriminate different

analytes

Ratio of retention factors

Primarily dependent on the nature of the analytes and theirinteraction with the stationary phase

Has to be >1


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Resolution

Ratio of distance between two peaks to the average width of

these peaks at baseline

Encompasses both efficiency and selectivity

Has to be 1.5 to be completely separated


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Efficiency: Plate theory

Column is considered to be divided into a number of

hypothetical plates

Each plate has finite height (height of effective theoretical plate,

HETP) and the analyte spends a finite time in this plate

Smaller plate height or greater number of plates, more efficient

separation


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Efficiency: Plate theory

Measure of chromatographic band broadening


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Sensitivity

Limits of detection

lowest quantity of a substance that can be distinguished from

the absence of that substance within a stated confidence limit

Limits of quantification

concentration at which quantitative results can be reportedwith a high degree of confidence

Signal to noise ratio: LOD 3, LOQ 10


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Calibration

Internal Standard Calibration

Standards must

have similar in analytical behaviour

not expected to be found in the samples

soluble in the same solvent/eluent

not a degradation product of the sample

not affected by target analytes, surrogates, or by matrix

interferences


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Calibration

Internal Standard Calibration

not as useful for GC and HPLC methods with non-MS

detectors, unless the internal standards could be separatedfrom target compounds chromatographically

for determination of caffeine, some internal standards used

are salicylic acid and nicotinic acid


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Calculation of Standards


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Peak Area vs Concentration


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Concentration of Samples


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Conclusions


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In this experiment, concentration levels of caffeine in tea,

coffee, and cola beverages were determined quantitatively by

HPLC

Obtain chromatogram: x-axis (retention time) and y-axis

(intensity of the response)

Peaks observed were the result of the sample run where the size

of the peak is proportional to the concentration of the analyte

response factor - concentration/area


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0.1892 M and 0.1587 M are the caffeine concentrationdetected for Great Taste, 0.008933 M and 0.009751 M

for Lipton, 0.003162 M and 0.003198 M for Pop, and

0.005174 M and 0.0005273 M for Zesto

Average values for both calculated values result a caffeine

concentration of 0.174 M for Great Taste, 9.342 mM forLipton, 3.18 mM for Pop, and 5.491 mM for Zesto


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END

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