HPLC( high performance liquid chromatography)

HPLC principle, machine, retention time ,application

1. HPLC
HIGH PERFORMANCE LIQUID CHROMATOGHAPHY
BY DR.AKHILESH KUMAR
3
RD
YR PG PATHOLOGY
DMCH DARBHANGA

2. How it Look

3. INTRODUCTION
►HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OR COMMONLY KNOWN AS HPLC IS AN
ANALYTICAL TECHNIQUE USED TO SEPARATE, IDENTIFY OR QUANTIFY EACH COMPONENT IN A
MIXTURE.
►THE MIXTURE IS SEPARATED USING THE BASIC PRINCIPLE OF COLUMN CHROMATOGRAPHY AND
THEN IDENTIFIED AND QUANTIFIED BY SPECTROSCOPY.
►HPLC IS THUS BASICALLY A HIGHLY IMPROVED FORM OF COLUMN LIQUID CHROMATOGRAPHY.
INSTEAD OF SOLVENT BEING ALLOWED TO DRIP THROUGH A COLUMN UNDER THE GRAVITY, IT IS
FORCED THROUGH UNDER HIGH PRESSURE OF UP TO 400 ATMOSPHERES

4. HPLC PRINCIPLE
►THE PRINCIPLE OF SEPERATION IN NORMAL PHASE MODE AND REVERSE
PHASE MODE IS ADSORPTION.
►WHEN A MIXTURE OF COMPONENT ARE INTRODUCED INTO A HPLC COLUMN,
THEY TRAVEL ACCORDING TO THEIR RELATIVE AFFINITY TOWARDS THE
STATIONARY PHASE.
►THE COMPONENT WHICH HAS MORE AFFINITY TOWARDS THE ADSORBENT,
TRAVELS SLOWER.
►THE COMPONENT WHICH HAS LESS AFFINITY TOWARDS THE STATIONARY
PHASE,TRAVELS FASTER.
►SINCE NO TWO COMPONENTS HAVE THE SAME AFFINITY TOWARDS THE
STATIONARY PHASE,THE COMPONENT SEPARATED.
►THE STATIONARY PHASE IS A GRANULAR MATERIAL WITH VERY SMALL
POROUS PARTICLES IN A SEPARATION COLUMN.

11. WHY USE HPLC
ADVANTAGE OF HPLC
►THIS IS AUTOMATED TECHNIQUE, SO LESS TIME IS REQUIRED
AND PERMITS PROCESSING OF LARGE BATCHES.
►VERY SMALL(0.5 micro liter of blood) SAMPLES ARE
SUFFICIENT FOR ANALYSIS,THIS IS SPECIALLY USEFUL FOR
PEDIATRIC WORK.
►QUANTIFICATION OF NORMAL AND ABNORMAL HEMOGLOBIN
VARIANTS CAN BE DONE ON EACH SAMPLE.
►THE METHOD IS HIGHLY SENSITIVE AND SPECIFIC.

12. DISADVANTAGE
►MORE COMPLEX –HIGHER SKILL LEVEL.
►EXPENSIVE
►CO-ELUTION OF Hb VARIANTS.
►TECHNICAL ASPECTS:CAREFUL EYE IS REQUIRED
►OTHER TECHNIUE MAY BE REQUIRED.

13. TWO IMPORTATANT TERM
RETENTION TIME-IT IS THE TIME IN MINUTES
MEASURE FROM THE TIME OF SAMPLE IJECTION TO
THE MAXIMUM POINT OF EACH PEAK OF NORMAL
HEMOGLOBIN AND ABNORMAL HEMOGLOBINS
RETENTION WINDOW-TIME IN WHICH THAT
PARTICULAR HEMOGLOBIN IS LIKELY TO APPEAR IN
THE ELUTE.

15. Important considerations for
interpretation
►ETHNICITY
►AGE
►CLINICAL COURSE:ASYMPTOMATIC/SYMPTOMATIC
►VARIANT HEMOGLOBIN PERCENTAGE
►A2 PERCENTAGE
►NUMBER OF VARIANTS
►CBC INDICES/RDW/PBS
►TRANSFUSION HISTORY
►GENETIC IMPLICATION

16. APPLICATION OF HPLC
THE HPLC HAS DEVELOPED INTO A UNIVERSALLY
APPLICABLE METHODE SO THAT IT FINDS ITS USE IN
ALMOST ALL AREA OF CHEMISTRY, BIOCHEMISTRY AND
PHARMACY
►ANALYSIS OF DRUGS
►ANALYSIS OF SYNTHETIC POLYMER
►FORENSIC TEST-DETERMINE OF STERIOD IN BLOOD URINE
AND SWEAT.
►ENVIRONMENTAL APPLICATION
►DETECTION OF PHENOLIC COMPOUNDS IN DRINKING
WATER
►BIO-MONITORING OF POLLUTANT.

17. THALASSEMIA AND HEMOGLOBINOPATHY CONDITION IN
INDIA
►ALFA CHAIN VARIANT
►G-PHILADELPHIA (ALFA68-ASPARAGINE TO LYSINE)
►BETA CHAIN VARIANTS
► S (BETA 6 GLU TO VAL)
► C (BETA 6 GLU TO LYS)
► E (BETA 26 GLU TO LYS)
►BETA THAL TRAIT/HOMOZYGOUS
►Hb D TRAIT/HOMOZYGOUS

18. ►Hb E TRAIT/HOMOZYGOUS
►Hb S Trait
►Hb D Iran/HOMOZYGOUS
►HPFH Trait
►Hb Lepore
►Hb Q India
►Hb S WITH HPFH

19. DIAGNOSIS ON HPLC
►WHAT ARE THE CBC AND PBS FINDING?
.ETHNICITY AND PLACE OF ORIGIN OF THE PATIENT
►ARE THE PEAK NORMAL (EXPECTED) OR IF THERE
A NEW PEAK
►WHERE IS THE PEAK LOCATED ?(IN RELATION TO
THE OTHER NORMAL EXPECTED PEAK)
►WHAT IS THE WIDTH & HEIGHT OF THE PEAK
►TOTAL AREA 1- 3 MILLION
►FAMILY SCREENING

20. HAMOGLOBIN CO-ELUTING WITH HbA2
►Hb Lepore
►Hb E
►Hb D iron

21. UNKNOWN PEAK
►May appear any where in the peak table.
►More than 1 unknown peak may be seen.
►Up to 8% not significant.
►If, above 8% - look for the RT for Hb identification
►For ex
►Unknown peak at RT 3.8+0.1 min is Hb D – Punjab
►If the area % is 22-40%, then it is Hb D – Punjab Heterozygous
►If the area % is 70-90%, then it is Hb D – Punjab Homozygous
►Unknown peak at RT 4.5+0.1 min is Hb Q india
►Area percentage is between 15 – 25 %

22. PEAK TABLE
►A1a –glycated from – normal upto 2.5%
►A1b – glycated from – normal upto 2.5%
►F – (refer next slide)
►LA1c/CHb – 1 – labile A1c – normal upto 4%
►LA1c/CHb – 2 – Carbamylated Hb – normal upto 4%
►A1c – changes with the gycemic status, <6% non diabetic level
►P3 – upto 6% acceptable
-6 to 12% may indicate sample deterioration
- 15 to 25% indicate Hb J

23. ►AO – non glycated fraction of Adult hemoglobin
►A2 – (refer next slide)
►S window – Sickle cell (Hbs)
- 22 to 40 % represent heterozygous condition
- 70 to 90 % represent homozygous condition
-(upto 10 % not significant )
►C window – Hb C
-22 to 40 % represent heterozygous condition
-70 to 90 % represent homozygous condition
- (upto 10 % not significant )
* Unknown-may appear anywhere in peak table/chromatogram

25. ANEMIA
Iron deficiency – A2 found to be slightly lower
Megaloblastic anemia – A2 found to be higher

26. CAUSES OF INCREASED / DECREASED HBA2 LEVELS……
Increased HbA2
►Beta Thalassemia
►Sickle cell anemia
►Megaloblastic anaemia
►Unstable
Hemoglobinopathies
►Hyperthyroidsm
Decreased HbA2
.G6PD deficiency
►Alpha Thalassemia
►HB Lepore
►Delta Beta thalassemia
►Delta thalassemia
►Alpha globin gene Variants

27. CAUSES OF ELEVATED HBF…..
►HBF – Major Hemoglobin of the Fetus & Newborn
►Can be 5 % for 12 – 24 months of age
►Increase in beta thalassemia and delta beta
thalassemia
►An increase in HBF in the 4
th
month of pregnanacy
(<5%)
►Increase in HBF in HPFH, Juvenile from of Chronic
Myeloid Leukemia
► (30 – 90 % HBF )
►Aplastic Anemia ( 5 – 15 % )

28. NORMAL PROFILE
►This profile usually indicates that there is no
evidence of thalassemia or hemoglobinopathy and
their carrier state with the following exceptions
►1) Mild alpha alpha – thalassemia mutations, such
as the common – alpha3.7 deletion
►Silent Beta – thalassemia alleles
►Thalassemia or unstable ALPHA – or BETA – chain
variants
►Hemoglobin variants that co- migrate or co-elute
with hemoglobin A

30. HBA2 MEASUREMENT
►It is a surrogate test used in the identification of
beta –thalassemia carriers
►Normal range 2.5 – 3.5 %
►In beta thalassemia carriers the HbA2 level is
usually higher than 4 %
►A number of beta thalassemia alleles have only a
minimally elevated HbA2
►Several conditions may reduce an originally
elevated HbA2

31. ►Beta thalassemia trait
►Clinically normal or mildly anemic .
►Hb normal or mildly reduced
►Microcytosis , Anisopoikilocytosis , target cells
►MCV <80 fl , MCH <27 pg,
►MCHC , RDW increased
►HbA2 - >3.6%, HbF - Normal

32. BETA THALASSEMIA
INTERMEDIA
Physical finding similar to
Beta Thal Major.
Patient is not dependent
on regular blood
transfusion for survival
Variable degree of anemia
Hypochromia
MCV,MCH markedly reduced
HbA usually below 10 – 35%
HbA2 low ,normal or
elevated
HbF raised

33. Beta thalassemia major
Severe anemia, ineffective
erythroiesis,iron overload,
extra medullary
hematopoiesis.
MCV,MCH markedly reduced
Marked RBC changes in
peripheral blood smear
HbA2 may be normal,low
or elevated
Marked reduction in HbA
HbF usually >85% (if HbF
>16.5% it may elute in
LA1c/CHb window

34. HB E TRAIT
Asymptomatic
Microcytosis with or without
mild anemia
May be confused with iron
deficiency
The red cell morphology may
be normal
Look for the following :
E elutes in the A2 window
For an E trait :
A2 will be between 25 –
35%
F will be normal

35. DOUBLE HETEROZYGOUS STATES
►Hb E/ Beta thalassemia
►Hb E/ S double heterozygous
►Hb E/ D double heterozygous

36. Hb E BETA THALASSEMIA
Look for the following :
Reduced indices
E elutes in the A2 window
For an E homozygous :
A2 will be between >50%
F will be elevated
These values will be variable
from patient to patient
Hb - <10gm/Dl
MCV - <70fL
Hb F - > 10%
Hb E - > 50%
SEVERITY-SEVERE

37. SICKLE CELL HETEROZYGOUS
Clinically asymptomatic
if not exposed to low O2
tension
Glutamic acid replaces
valine at 6
th
position of
Beta chain
When exposed to low O2
tension when lead to
Vaso-occulisive crises.
Normocytic
Normochromic RBC
HbS ranges between
30-40 %

38. HbS HOMOZYGOUS
Chronic haemolytic anaemia
Normal Hb replaced by sickle
Hb
Sickle shaped RBC
Anaemia moderate to severe
(Hb 4 – 8 g/dL)
ISC 5 -10% of RBC in peripheral
smear
HbA2 normal or slightly
elevated
HbF 15 -25% in central india
HbF 11 – 20% in odisa
HbS around 70 to 90% of total
Hb

39. Double Heterozygous for HbS and HPFH
Asymptomatic or mild
sickle cell disease
Normocytic
Normochromic with
irreversibly sickled cells
Hb Electrophoresis
shows SFA2 pattern
HbF ranges between 20
-30%
HbS around 60%
HbA2 usually normal
(<3%)

40. HbD-Punjab
Heterozygous
Hb D: beta 121 Glutamic
acid replaces Glutamine
Normal or Asymptomatic
Prevalence of 3% in
North India
HbD Punjab elutes in an
unknown window at a
retention time around
3.8 mins
Abnormal Hb ranges
between 33 – 43%

41. heterozygous for
HbD Punjab and
beta Thal trait
Asymptomatic carrier with
mild anemia similar to Beta
Thal Trait
Mainly found in Punjabis and
north Indians
MCV, MCH low, raised RBC
count
Unknown peak around 3.8
min RT approx 80 – 90% of
total Hb
Mild elevated HbF(3-6%)

42. Double Heterozygous for HbS
& HbE
Asymptomatic , mild
anaemia
Sickling crisis is
uncommon but may
happen during pregnancy
and air travel
Microcytosis , target
cells are common , sickle
cells are rare
Mild reticulocytosis
Both HbS & HbA2 are
>30% HbF mildly elevated

43. Double Heterozygous for HbS
& HbD
Although individually both HbS &
HbD traits are asymptomatic but
this condition results in
moderately severe sickle cell
anemia
Found mainly in Punjabi Muslims
Beta121 glutamine residue
increases the polymerization of
HbS
Chronic mild to moderate anemia
with increased reticulocytes
Hb around 6 -10 gm/dL
Separate peaks in D and S window
HbF around 10 – 20%

44. Hb Q India Heterozygous
Asymptomatic
Mutation in the Alpha 1 gene,
GAC->CAC at codon 64
A few incidents detected in
Goa and Sindhi families of
Mumbai
Unknown peak around 4.45
min RT in D10 system and
4.77 min RT in Variant
system
Constitutes around 8.7 –
23.3% of total Hb

45. HbH
Mild to chronic haemolytic
anaemia
Defect in Alpha chain synthesis
results in accumulation of Y and
Beta chains
Tetramers of Beta chains (HbH)
are unstable
They have high O2 affinity and
useless for O2 transport
Oxidise easily and precipitated
with cell ageing
On D10 system significant peak
appear in the first mins of elution
suggestive of a thal / HbH
Inclusion body test or DNA studies
required for confirmation

46. Hb Bart’s Newbron
Defect in alpha chain
synthesis
Tetramers of alpha chains
At birth 20 – 40 % Hb Bart
“s is foun
Low HbA2
Significantly at first mins
of elution

47. Hb Lepore Trait
Unequal crossover during
meiosis with deletion of 3 part
of delta and 5 part of Beta gene
leading to delta –beta fusion
gene
Sean in Italian , Greek & African
In India found in Agarwal &
Mehtas
Anemia and reticulocytosis
Coelutes in HbA2 window
In D10 system it elutes as an
unknown peak around 1.85 min
RT

48. DOUBLE HETEROZYGOUS FOR HbS
AND BETA THAL TRAIT
Clinical manifestations resemble
sickle cell anaemia and persistent
splenomegaly
Laboratory parameters of both sickle
cell anaemia & thalassemia are
observed
Target cells predominate blood picture
Reduced MCV ,MCH ,MCHC
Hb Electrophoresis shows SFA2
pattern
HbS around 90%
HbA2 around 5% , HbF around 6.5%