chromatography column liquid chromatography HPLC technique

1. Presenter: Dr.ANURAG YADAV
Moderator: Dr.AVINASH.S.S

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

3. 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.

4. 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.

5. • 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

6. Components of HPLC
1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Computer
7. Waste collector

9. 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

11. 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

12. 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.

13. Pumps in HPLC

14. 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

16. 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

17. 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.

18. 18
Manual Injectors
Front View
Inject
Rear View
Load - Inject
Sample Loop

19. 19
Automatic Injectors
Step 1 Step 2
Step 3

20. • 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

21. 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.

22. 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

23. 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.

24. Several Column Types
( can be classified as)
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange

25. 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

26. 26
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.
Polar Group
silica

27. 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

28. 28
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.
C18 phase
silica
Si
Me
Me
O Si
O
O

29. 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.

30. 30
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)

31. 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.

32. 32
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 support
and are later eluted with acid (H+)
Cations exchange Na+ silica
S
O
O
ONa

33. 33
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 support
and are later eluted with acid (H+)
Anions exchange Cl- silica
Me N
Me
Me
CH2
Cl

34. HPLC Columns
Within 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.

35. 35
Modes of High Performance Liquid
Chromatography
Types of Compounds Mode Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
Phase
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids Ion
Pair
C-18, C-8 Water/Organic
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Phase
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions Ion
Exchange
Anion or Cation
Exchange
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel Filtration-
Aqueous
Gel Permeation-
Organic

36. 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]

37. 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

38. 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

39. 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

40. 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.

41. • 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

42. 42
Common HPLC Detectors
•UV-VIS
•Diode Array
•Multiple Wavelength
•Variable Wavelength
•Mass Spectrometers
•Refractive Index
•Fluorescence
•Light Scattering
•Electrochemical
•Radioactivity
•Conductivity

43. 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.,

44. 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.

45. 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.

46. 4 .Electrochemical detector
• Electroactive analytes
• Electrochemically measures
 Oxidation reaction: hydrocarbon,
amines, phenols, catecholamines
 Reduction reaction: alkenes,
esters, ketones
• Detection limit: pg to ng.

47. 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.

48. 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.

49. HPLC Detectors

50. 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.

51. 51
How can We Analyze the Sample?
For example:
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose
1
2
3
4
5
mAU
time
6

52. 52
Separations
Separation 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

53. 53
SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

54. 54
SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

55. 55
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

56. 56
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

57. 57
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

58. 58
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

59. 59
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

60. 60
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

61. 61
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

62. 62
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

63. 63
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

64. 64
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

65. 65
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

66. 66
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

67. 67
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

68. 68
SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

69. 69
The Chromatogram
Injection
to
tR
mAU
time
tR
to - elution time of unretained peak
tR- retention time - determines sample identity
Area or height is proportional
to the quantity of analyte.

70. Preparation for HPLC
Sample 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

71. 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

72. 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

73. 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.

74. Acceptance criteria
0.05ml for small quantities
0.1ml 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

75. 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

76. 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 injections
M=average area
X=area
For system precision % of RSD is not more than 1

77. 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

78. 5.Detector wavelength accuracy
Column : C18
Mobile phase: HPLC grade methanal
Flow rate:1ml/min
Retention time:5 minutes
Wavelength: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

79. 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

80. 2. PUMP
Possible 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

81. 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 samples
3.Sterilize when fresh sample is
used

82. 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

83. 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

84. 6.Software
Update frequently , around 6-12 months

85. TROUBLESHOOTING
Identifying problem using a chromatogram
1.The above pattern occurs when the mobile phase is not
suitable

86. 2.Baseline noise
I. Blank base(normal)
II. Noisy :
•occurs when there is
contamination of
mobile phase
• improper cleaning

87. 3.Drift
•Temperature is
unstable
•Contamination of
column

88. 4.Peak tailing
•Improper mixing of mobile phase(90% of the time)
•Problems with column(30% of the time)

89. After adjusting PH with buffer
Good resolution

90. 5.Peak fronting
•Low temperature
•Overloading of sample
•Flow rate
•Overfilling of column
•Insufficient mixing of mobile phase

91. 6.High concentration of sample

92. RT=4.0
7.Retention time variation
Occurs due to problems with
mobile phase
RT=3.
0
RT=3.5

93. 8.Communication error
Due to problem in communication between detector and the software

94. 9.Peak splitting
Occurs when column lifetime is diminished

95. 10.Power fluctuation

96. 11.Variation in area
•Problem with injector
•Fluctuation in flow rate
•Error in detection
A=3010
A=3215
A=3516

97. 12. Ghost peaks
•Contamination of mobile phase
•Upset equilibrium
•Production not finished

98. 13.Detector lifetime diminished

99. Applications of HPLC
Therapeutic & 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.

100. Applications of HPLC
Therapeutic & diagnostic uses
• Measurement of drugs & other
metabolites in biological fluids
• Monitoring of cirrhosis pt through
aquaporin-2 in urine

101. 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

102. 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.

103. Advantages of HPLC
1. Needs a small sample with a high accuracy and precision
2. Non-destruction of sample

104. Disadvantages
• Need a skill to run the instruments
• Solvents consuming
• Maintenance .

105. 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

106. 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.

107. • 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.

109. 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.

110. • The particles are made of polystyrene-divinylbenzene &
are available under the name of POROS.

111. References
• Keith wilson-technique text book.
• Tietz –clinical chemistry text book.
• Kaplan-technique text book.
• Upadhyay- biophysical chemistry.

114. 114
UV-Vis Detectors
b
c
Detector Flow Cell
I0 I
Log I0 = A = abc
I
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 ).

115. 115
Fluorescence Detection
Emission
Monochromator
signal&
spectramode
PMTdetector
ReferenceDiode
8µlFlowCell,auto-recognition
Triggerpack
Exitation
Monochromator,
signal&
spectramode
Mirror
Lens
(condensorEX)
Lens(condensorEM)
SlitEM SlitPMT
SlitEX
Diffuser
Xenon
flashLamp,
15W