chromatography hplc iii-anu

Presenter: Dr.ANURAG YADAVModerator: Dr.AVINASH.S.S

Chromatography-III

HPLC

Contents

• HPLC:– Definition – Basic principle– Instrumentation– Practical consideration – Applications – Advantages & disadvantages of HPLC

HPLC (High-performance liquid chromatography)

• Type of liquid chromatography

• Conducted in a column

• Characterized by the use of high pressure & small particle

size to push a mobile phase solution through a column of

stationary phase allowing separation of complex mixtures

with high resolution.

Basic principle

• Based on distribution of solute between a liquid mobile phase

and a stationary phase.

• The small diameter particles are used as stationary phase

support.

• The table shows relation between various parameters of HPLC.

• Trendline:

• Stationary phase have small particulate size and high surface areas.

• Columns: 20 cm or less• Mobile phase pumped at high pressures of

400Bar, 6000 psi. • Flow rates: 1-3 cm3 per min

Column length No. of theoretical plates per unit area

Resolving power Column length

Particle size Surface area

Basic principle

Components of HPLC

1. Solvent Reservoir

2. Pumps

3. Sample Injection System

4. Columns

5. Detectors

6. Computer

7. Waste collector

Solvent Reservoir (mobile phase)

• Inert container → mobile phase • Must be free of contaminants & bubble forming gases that get

trapped in column or detector.• Measures: microfilter, degasser• Degassing : - Vacuum filtration - Warming - Stirring vigorously with magnetic stirrer

- Sparge with inert gas (N2 or He)

- Ultrasonication

HPLC Pump

• To produce an appropriate pressure to push solvent & sample into

the column.

• Ideal pump

Deliver high pressure (upto 50MPa)

Deliver pulse free flow

Constant volume delivery

Deliver high volumes (flow rates) of solvent (to 10 mL/min)

Solvent replacement is easy

Types of HPLC Pump

Constant Pressure

• A steady pump pressure

• (usually about 1000–2000 psi) is needed to ensure

reproducibility & accuracy.

Constant displacement Pump

Reciprocating pump: constant flow rate through the column

Slight cyclical variation in pressure→pulse dampeners.

Pumps in HPLC

HPLC Pump operating mode

Isocratic elution: A separation that employs a single solvent or solvent mixture of constant composition with single pump throughout the run.

Uses: Simple separation & compound with similar structures & retention time

Gradient elution: Here two or more solvent systems that differ significantly in polarity with separate pumps.

the ratio of the solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. Separation efficiency is greatly enhanced by gradient elution.

Uses : complex separations

Sample Injector

1) Manual Injector:• Manually load sample into the injector using a syringe• and then injected sample → into the flowing mobile phase

→which transports the sample into the beginning (head) of the column, which is at high pressure

Sample Injector

2) Autosampler injector:

• User loads vials filled with sample

solution into the autosampler tray

(100 samples)

• Autosampler automatically

Takes appropriate sample volume,

injects the sample,

then flushes the injector to be

ready for the next sample, until all

sample vials are processed.

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Manual Injectors

Front View

Inject

Rear View

Load - Inject

Sample Loop

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Automatic Injectors

Step 1 Step 2

Step 3

• Control of column temperature as constant temp is required.

• Achieved by:– Column chambers– Water jackets– Temperature controlled blankets– Heating/cooling blooks

• Stable column temperature is required to generate reproducible retention time.

Column Heater/Chillers

Guard column

• Repeated application of impure samples (sera, urine) →

↓column resolving power.

• Installed between injector & analyte column

• Column characterstics: short(1-2cm), same ID & packed with

similar particles to that present in analytical column.

• Retains contaminating particles, can be replaced at regular

intervals.

Column:

• Considered the “heart of the chromatograph” the column’s stationary

phase separates the sample components of interest using various physical

and chemical parameters.

•The small particles inside the column are what cause the high

back pressure at normal flow rates.

•The pump must push hard to move the mobile phase through the

column and this resistance causes a high pressure within the

chromatograph.

Analyte Columns in HPLC

Analyte Columns in HPLC

• Standard Column : 3-25cm long, ID(4.6 mm) ;optimum flow volume = 1 ml/min.

• Narrow Bore : 3 mm(ID); flow volume 0.6 ml/min • Microbore /Open tubular : 25-50cm long, 1 mm(ID); 50

microliter/min.

Several Column Types ( can be classified as)

• Normal phase

• Reverse phase

• Size exclusion

• Ion exchange

Normal phase • In this column type, the retention is governed by

the interaction of the polar parts of the stationary phase and solute.

• For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

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HO Si

O

O

STATIONARY PHASES(NORMAL POLARITY)

Silica or alumina possess polar sites that interact with polar molecules.

Most polar…….Least polar

Components elute in increasing order of polarity.

Components elute in increasing order of polarity.

Polar Group

silica

Reverse phase• In this column the packing material is relatively nonpolar and the solvent is

polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase.

• Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

Common Reverse Phase Solvents –

Methanol

Acetonitrile

Tetrahydrofuran

Water

CH3OH

CH3CN

H2O

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STATIONARY PHASES(REVERSE POLARITY)

If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules.

Most non-polar…….Least non-polar

Components elute in decreasing order of polarity.

Components elute in decreasing order of polarity.

C18 phasesilica

Si

Me

Me

O Si

O

O

Size exclusion

• In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size.

• Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

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STATIONARY PHASES(SIZE EXCLUSION)

Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minium size.

Larger molecules…….Smaller molecules

Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)

Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)

Ion exchange

• In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase.

• Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

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STATIONARY PHASES(CATION EXCHANGE)

Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged)

Most +ve…….Least +ve

+ve charged species adhere to the supportand are later eluted with acid (H+)

+ve charged species adhere to the supportand are later eluted with acid (H+)

Cations exchange Na+ silica

S

O

O

ONa

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STATIONARY PHASES(ANION EXCHANGE)

Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged)

Most -ve…….Least -ve

-ve charged species adhere to the supportand are later eluted with acid (H+)

-ve charged species adhere to the supportand are later eluted with acid (H+)

Anions exchange Cl- silica

Me N

Me

MeCH2Cl

HPLC ColumnsWithin the Column is where separation occurs.

Key Point –Proper choice of column is critical for success in HPLC

Materials of construction for the tubing• Stainless steel (the most popular; gives high pressure capabilities)• Glass (mostly for biomolecules)• PEEK polymer (biocompatible and chemically inert to most solvents

Packing material:

The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN.

Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material.

According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.

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Modes of High Performance Liquid Chromatography

Types of Compounds Mode StationaryPhase

Mobile Phase

NeutralsWeak AcidsWeak Bases

ReversedPhase

C18, C8, C4cyano, amino

Water/Organic Modifiers

Ionics, Bases, Acids Ion Pair

C-18, C-8 Water/Organic Ion-Pair Reagent

Compounds notsoluble in water

NormalPhase

Silica, Amino,Cyano, Diol

Organics

Ionics Inorganic Ions Ion Exchange

Anion or CationExchange Resin

Aqueous/Buffer Counter Ion

High Molecular WeightCompoundsPolymers

Size Exclusion

Polystyrene Silica

Gel Filtration- AqueousGel Permeation-Organic

Types of columns in HPLC:

• Guard Column

• Fast Column

• Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)

• Capillary(i.d. 0.1 -1.0 mm; various lengths)

• Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)

• Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]

Fast Column

• One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time).

• Fast column are designed to decrease the time of chromatographic analysis

• Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm diameter.

• Advantages-

Increased sensitivity

Decreased analysis time

Decreased mobile phase usage

Increase reproducibility

Capillary Column

• It is also known as micro columns

• It has a diameter much less than a millimeter and there 3 types:

Open tubular

Partially packed

Tightly packed

They allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness

Preparatory Column

• Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application.

• It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system

Column packing

• Particulate packings : particle diameters 1.8 to 10 μm.

1) Bonded phase : stationary phase is bonded chemically to

surface of silica particles through a silica ester or silicone

polymeric linkage.

Eg. In reverse phase HPLC, ODS bound to silica particles

(C 18 column).

2) Polymeric : eg. Polystyrene-polyvinylbenzene, stable at PH

2-13

3) Chiral : separation of enantiomers.

• The detector can see (detect) the individual molecules that come

out (elute) from the column.

•A detector serves to measure the amount of those molecules

so that the chemist can quantitatively analyze the sample

components.

•The detector provides an output to a recorder or computer

that results in the liquid chromatogram(i.e., the graph of the

detector response).

Detectors in HPLC

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Common HPLC Detectors

•UV-VIS•Diode Array•Multiple Wavelength•Variable Wavelength

•Mass Spectrometers

•Refractive Index

•Fluorescence

•Light Scattering

•Electrochemical

•Radioactivity

•Conductivity

1. Variable wavelength (UV-VIS absorbance) detector

• UV-Visible spectrometry

• Measures absorbance of light as

down to 190nm.

• Detection limit < 1 ng

ADV:

• High sensitive

• Small amount of sample.

Uses: amides, carboxylic acid,

cholesterol.,

2. Scanning wavelength detector

• Records complete absorption spectrum of analyte

• By using photodiode array technique

• Scans complete spectrum of analyte within 0.01 sec

• Adv : could analyse sample simultaneously at diff wavelength.

• Disadv: less sensitive than UV-VIS detector.

3. Fluorescence detector

• Based on measurement of fluorescence

• Compared to UV-Vis detectors fluorescence detectors offer a higher

sensitivity and selectivity that allows to quantify and identify

compounds in complex matrices at extremely low concentration levels

(trace level analysis).

• Detection limit: pg to ng

• Uses: amino acids and amines.

• Limitation: only fluorescent

• analytes measured.

4 .Electrochemical detector

• Electroactive analytes

• Electrochemically measures

Oxidation reaction: hydrocarbon,

amines, phenols, catecholamines

Reduction reaction: alkenes,

esters, ketones

• Detection limit: pg to ng.

5. Refractive Index (RI) Detection

• The refractive index (RI) detector uses a monochromator and is one of the least sensitive LC detectors.

• This detector is extremely useful for detecting those compounds that are non-ionic, do not absorb ultraviolet light and do not fluoresce.

• e.g. sugar, alcohol, fatty acid and polymers.

LC-MS• HPLC column eluted solutes

introduced into a ion source of a MS, blasted with electrons, which cause them to turn into positively charged molecular ions and fragmented ions (ion source).

• When these charged particles passed through filter → separated according to m/e ratio → ions collected.

• TIC the current generated by all such ions from analytes is measured, which would be proportional to the concentration of analyte.

HPLC Detectors

Computer

• Electronic signals generated by detectors are recorded in the

form of chromatograghic peak at varied function of time

• Peak Area, height, retention time, base width of

chromatograghic peak is measured to compute analyte

concentration of each peak.

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How can We Analyze the Sample?

For example:Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose

1

23

4

5

mAU

time

6

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SeparationsSeparation in based upon differential migration between the stationary and mobile phases.

Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica

Mobile Phase - carries the sample through the stationary phase as it moves through the column.

Injector

Detector

Column

Solvents

Mixer

Pumps

High Performance Liquid Chromatograph

Waste

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SeparationsInjector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

High Performance Liquid Chromatograph

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SeparationsInjector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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The Chromatogram

Injection

to

tR

mAU

time

tR

to - elution time of unretained peak

tR- retention time - determines sample identity

Area or height is proportionalto the quantity of analyte.

Preparation for HPLCSample preparation :

• Sample purification and Sample Derivatization.

• Before HPLC analysis , solutes are chemically derivatized

before or after chromatographic separation to increase their

ability to be detected .

• Eg. Derivatization of amino acids with O-Phthalaldehyde OR

Dansyl chloride.

• Degassing

Factors which influence the HPLC performance

1. Internal diameter of column

- the smaller in diameter, the higher in sensitivity

2. Pump pressure

- the higher in pressure, the higher in separation

3. Sample size

4. The polarity of sample, solvent.

5. Temperature

- the higher in temperature, the higher in separation

CALIBRATION

Calibration of HPLC is done to check the performance of its instrument.

1.flowrate(pump calibration)

2. Detector and injector linearity

3.System precision

4.Column oven temperature

5.Detector wavelength accuracy

1.Pump calibration

• Disconnect the column and connect the inlet and outlet tubing’s with a union.

• Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles.

• Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighed beaker and collect the mobile phase for 10 min. weigh the beaker to get the weight of mobile phase.

• Calculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time).

• Calculate the corresponding flow rate. Carry out the experiment in duplicate.

Acceptance criteria

0.05ml for small quantities0.1ml for larger quantities

2.Detector and Injector linearity

Column : ODS or C18

Mobile phase : milli Q water and acetonitrile (80:20)

Flow rate: 1ml/min

Temperature: 40°Centigrade

Detector wavelength:272 nm

Runtime:10min

Preparation of stock solution:

•Take 50mg of caffeine and transfer into 50ml of flask and make it up to the mark with the diluent

•Caffeine is used a it gives a multiwavelength response and is stable

•Prepare solutions of different PPM (100-600)

•Inject them and calculate the area

•Correlation coefficient r is used to check the detector linearity and cannot be less than .999

• r = NƩXY-ƩXƩY/√ [NƩX²-(Ʃx) ²][NƩY²-(ƩY)²]

•The graph obtained between concentration and area is linear

3.System precision

•From the stock solution pipette out 1ml of stock solution and transfer into 10ml flask and make it upto the mark with diluent for 100PPM conc.

•Calculate the percentage of RSD of areas of 5 different injections

•% RSD = standard deviation/average value of above 5 injection areas × 100

•Where SD = √ Ʃ(X-M) ²/n-1

N= # of injectionsM=average areaX=area

For system precision % of RSD is not more than 1

4.Column oven temperature

1. Set the column oven temperature to 30° and leave it for 30 minutes

2. Open the door of the column oven and keep the thermometer and leave it for 30 min

3. now note down the reading in the thermometer

4. Similarly change the column oven temperature to 40°C and 50°C and repeat the above procedure

Acceptance criteria

±2°c

5.Detector wavelength accuracy

Column : C18

Mobile phase: HPLC grade methanalFlow rate:1ml/minRetention time:5 minutesWavelength:272nm

1. Inject the methanol and record blank

2. Inject 20 ml of standard solution at 268nm

3. Similarly inject the standard solution by increasing the nm upto 278nm(increasing it 1 at a time)

Acceptance criteria

273nm ± 1

MAINTAINANCE

1.RESERVOIR

Possible Cause Preventive Maintenance

1. Blocked inlet frit 1. a. Replace (3–6 months)

  b. Filter mobile phase with 0.4 - 0.5 µm filter

2. Gas bubbles 2. Degas mobile phase, sonification

2. PUMPPossible Cause Preventive Maintenance

1. Air bubbles 1. Degas mobile phase , do not change mobile phase during run

2. Pump seal failure 2. Replace (3 months),clean with 1 N acid

3. Check valve failure 3. Filter mobile phase; use inlet-line frit ; keep spare

 4.Improper cleaning 4.Clean with Isopropyl alcohol , mobile phase container must be cleaned with mobile phase and other sections with solvent

3.INJECTOR

Possible Cause Preventive Maintenance

1.Washing 1.wash before and after use

2. Rotor seal wear 2. a. Do not overtighten

 3.Syringe

b. Filter samples3.Sterilize when fresh sample is used

C. Injector

4.Column

Possible Cause Preventive Maintenance

1. Number of injections 1. 2000 or less

2. Blocked frit 2.. a. Filter mobile phase

  b. Filter samples

  c. Use in-line filter and/or guard column

3. Void at head of column 3.. a. Avoid mobile phase pH >8

  b. Use guard column

  c. Use precolumn (saturator column

D. Column

5.Detector

Possible Cause Preventive Maintenance

1. Lamp failure; decreased detector

1. Replace (6 months) or keep spare lamp

  response; increased detector noise

2. Bubbles in cell 2. a. Keep cell clean

  b. Use restrictor after cell

  c. Degas mobile phase

6.Software

Update frequently , around 6-12 months

TROUBLESHOOTING

Identifying problem using a chromatogram

1.The above pattern occurs when the mobile phase is not suitable

2.Baseline noise

I. Blank base(normal)

II. Noisy : •occurs when there is contamination of mobile phase• improper cleaning

3.Drift

•Temperature is unstable•Contamination of column

4.Peak tailing

•Improper mixing of mobile phase(90% of the time)

•Problems with column(30% of the time)

After adjusting PH with buffer

Good resolution

5.Peak fronting

•Low temperature•Overloading of sample•Flow rate•Overfilling of column•Insufficient mixing of mobile phase

6.High concentration of sample

RT=4.0

7.Retention time variation

Occurs due to problems with mobile phase

RT=3.0

RT=3.5

8.Communication error

Due to problem in communication between detector and the software

9.Peak splitting

Occurs when column lifetime is diminished

10.Power fluctuation

11.Variation in area

•Problem with injector•Fluctuation in flow rate•Error in detection

A=3010

A=3215

A=3516

12. Ghost peaks

•Contamination of mobile phase•Upset equilibrium•Production not finished

13.Detector lifetime diminished

Applications of HPLCTherapeutic & diagnostic uses

In clinical diagnosis : detection and

estimation of amino acids,

metabolites, sugars in physiological

samples,

Mucopolysaccharides in urine and

blood –useful for screening and

diagnosis of inborn metabolic

disorders

Clinical diagnosis of Aminoacidurias,

hemoglobinopathies,

mucopolysaccharidoses, etc.

Separation & identificaton of lipids,

carbohydrates & proteins.

Applications of HPLC

Therapeutic & diagnostic uses

• Measurement of drugs & other

metabolites in biological fluids

• Monitoring of cirrhosis pt through

aquaporin-2 in urine

Applications of HPLC

Forensic analysis• Forensic analysis of blood and

urine alcohol levels• Forensic analysis of blood and

urine levels of drug abuse and steroids.

• Used for toxiclogical analysis of biological fluid – op poisoning

Applications of HPLC

Pharmaceuticals industry uses• Quantity of drug determination from pharmaceutical dosage

forms, ex. Paracetamol determination in panadol tablet• Quantity of drug determination from biological fluids.

Analysis of natural contamination

- Phenol & Mercury from sea water

Food and essence manufacture

- sweetener analysis in the fruit juice

- preservative analysis in sausage.

Advantages of HPLC

1. Needs a small sample with a high accuracy and precision

2. Non-destruction of sample

Disadvantages

• Need a skill to run the instruments

• Solvents consuming

• Maintenance .

Advantages & disadvantages

HPLC

• Both volatile & non-volitile

analytes can be measured

without degradation.

• Time of separation is very less

bec high pressure pump is used.

• Length of column is small

• Non-explosive

• Non-expensive

GC

• Only volatile analytes can be

measured.

• More time

• Bigger coloumn than HPLC

• Explosive

• Expensive

Ultra-performance liquid chromatography (UPLC)

• As pointed earlier, the resolution of a mixture of analytes

increases as the particle size of stationary phase decreased.

• But such a decrease leads to a high back-pressure from the

eluent flow.

• Solution to this is represented by HPLC with new sationary

phase less than 2μm diameter by the waters corporation (1.7 μm

diameter)- made of Bridged Ethylsiloxane silica Hybrid (BEH)TM

• This can sustain of back-pressure of 150MPa.

• The instrument available under the trade name of

ACQUITYTM & the term UPLC is registered under

Waters.

• this operates upto 10 times faster than the conventional

HPLC, & can complete in less than 5mins

• The peaks may last for only 1s, so the detectors should

respond ultra fast to respond to the peaks.

Perfusion chromatography

• The high resolution achieved by the HPLC si based on use of

small diameter particles.

• However, same can be achieved at low cost by generating high

flow rate without high pressure.

• Perfusion chromatography comes with this, with particle size

around 10-50μm diameter, that have channels of around 1μm

diameter running through them.

• The high flow rate result in small plate heights & hence high

resolution in very short separation times.

• The particles are made of polystyrene-divinylbenzene &

are available under the name of POROS.

References

• Keith wilson-technique text book.

• Tietz –clinical chemistry text book.

• Kaplan-technique text book.

• Upadhyay- biophysical chemistry.

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UV-Vis Detectors

b

c

Detector Flow Cell

I0 I

Log I0 = A = abcI

Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law.

Characteristics: Specific, Concentration Sensitive, good stability, gradient capability.

Special: UV-Vis Spectral capability (Diode Array Technology ).

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Fluorescence Detection

Emission Monochromatorsignal & spectra mode

PMT detector

Reference Diode

8 µl Flow Cell, auto-recognition

Trigger pack

Exitation Monochromator,signal & spectra mode

Mirror

Lens(condensor EX)

Lens (condensor EM)

Slit EM Slit PMTSlit EX

Diffuser

Xenonflash Lamp,15 W