1.

In my analysis by HPLC,In sample main peak and placebo peak are

eluting with shorter resolution ( almost look like doublet). how can i get
max resolution ? Is changing gradient method is usefull?

You can try one or more of the following:

1) Changing the column type (c8 to c18, etc.)

2) Changing the Mobile phase composition (increase buffer

etc.)

3) Decrease the particle size on column (from 5 to 4, 3

etc.)

4) Increase the column length

5) Decrease the flow rate

6) If all of the above fails, then as you suggested,

gradient method can be used to separate the two peaks.

P.S. : Gradient method can also be used as an alternative

to the above methods and not necessarily the ultimate
solution.

2.Why Deuterium lamp is used as UV light Source for UV.

deuterium can be operated in 117nm to 900 nm all coloured

compounds are operated at this range

3.full name of OOS & OOT ? Dercribe it with details ? why this is needed in
pharma industry ?

OOS stands for out of specification, if any batch in plant

doesn't meet the desired specifications because of various
reasons then concern QC will raise out of specification
base on the results, based on the out time OOS we can
perform use test so as to qualify material for next step

OOS can relased before releasing complete intermediate

report
oot for occur when a stability result does not follow the
expected trend, either in comparison with other stability
batches or with respect to previous results collected
during a stability study.

4.what is the difference between BDS and ODS Columens and both are
same or not

BDS means base deactivated Silanol through Special

teachnique residual silanols deacivated and silanol
activity reduced it is endcapped column.it is good for
basic compounds. ODS means octa decyl silane it is normal
column.high acidic silica it is not suitable for basic
compounds.
5.DIFFERENCE BETWEEN C18 & C8 COLOUMN ? WHY C18 COLOUMN WIDE
USE METHOD DEVELOPEMENT. ?

the difference between these two columns is packing only

C18- octa decyl chain linked to silica.
c8- only octyl chain linked to silica.

C18 is widely used, the polymeric C18 format incorporates a

tri-functional silylation procedure whereby the octadecyl
group is bonded to 2 or 3 silica atoms on the silica gel
backbone. This increased silylation results in far greater
column stability particularly in acidic mobile phase
conditions. Stereo recognition capability is also greater
than that of the monofunctional silylation type of C18.

6.Why caffeine as a reference standard used for calibration of HPLC?

Because of

# It gives a multiwavelength response like 205,245,273 nm

# It is easily available in market
# It is very stable, we can use a long time for analysis
i.e. it is not degradable
# It is chip
7.WHAT ARE THE LIMITS FOR UNIFORMITY DOSAGE CONTENT FOR TABLETS
AND CAPSULES

Uniformity of dosage units are determined from two ways.

1.Content Uniformity
2.Weight variation

Content Uniformity is applied when tablet or capsules having

25mg or below and
when active pharmaceutical ingradient 25% weight present in
the total tablet weight.

weight variation test is versa.

Acceptance critiria for uniformity of Dosage units are for

content uniformity Acceptance value cannot be more than 15.

calculation of acceptance value is

A.V=(X-M)+K.S
where X=Mean value of 10 units
M=98.5 or 101.5
K=2.4(constant value) for 10 units
2.0(constant value) for 30 units
S=Standard Deviation value

in the above expression there are three cases

a)A.V=K.S(when X value lies 98.5 to 101.5)
b)A.V=(X-M)+K.S(when X value is more than 101.5)
c)A.V=(M-X)+K.S(when X value is less than 101.5)

8.What is Fourier Transform Spectrosopy?

This is a family of spectroscopic techniques in which the

sample is irradiated by all relevant wavelengths
simultaneously for a short period of time.

The absorption spectrum is obtained by applying a

mathematical analysis to the resulting energy pattern.
9.what is the significant of pH in HPlc?

2-7.5
9.IF HPLC CALIBRATION FAILED THEN WHAT YOU DO

ONCE U CAN CHECK THE PREVIOUS ANALYSIS AND IT IS IMPURITY

PROFILE AND THAT BATCH MUST BE COMMERCIAL AND ANALYSED TAHT
SAMPLE U CAN CHECK IT OUT PREVIOUS IMPURITY PROFILE BOTH
PROFILES ARE SAME THAT IS NO PROBLEM NOT MATCHED CAL THE
SERVICE ENGENEER.INFORM THE CUSTOMER......
10. Why we are using only KBr in IR spectrometer why not KCl

KBr shows Absorbance in 2.4 -16 MICROMETER of IR REGION.

While other compound shows Transmittance in 2.4 -16
MICROMETER of IR REGION. & hence resolution is not achieved .

& for the purpose of resolution & background correction KBr

is the best option.

10.what is the defination of calibration & verification?

Definitions:
------------

Calibration: This is an act of checking or adjusting by

comparing with known standard for accuacy of the instrument
to meet the defined criteria with a specificed degree of
confidence.

Performance Verification:
-------------------------

It is an activity to document that the instrument

consistently performs accordingly to the predefined
specifications which is appropriate for intended use.

Differences:
------------

Calibration is the process where we can do adjustment is a

part of calibration but not necessary.

Where as Peformance Verification is only the act to check

the Performance of the instrument.

Example
-------

Using Salicylic acid and Prednisone Tablets for Dissolution

Apparatus (Chemical testing) is a Performance Verification
test rather being called as "Calibration" since we are
checking the performance of the equipment whereas we are
not doining any adjustment at any point of time .Hence it
is eventually called as "Performance Verification"
11. What is the difference between Chromatograhic Purity And Related
substances

Related substance tests are established after complete

impurity profiling of respective substane where as
Chromatographic purity demonstarte the actual amount of the
substance present with unknown impurities.
12.what is the difference betwen the analytical column & peparative
column?

Column dimension (diameter) is different . In preparative

column we can give high flow rate (depends upon the
diameter)

Preparative column :9.4mm*250 5micron

Analytical column :4.6*250mm, 5micron

13.what is method development parameter in HPLC method development

Parameters involved during initial stage of Analytical

Method development:

1. Optimization of Mobile Phase

2. Selection of Buffers
3. Selection of Ion pair reagent
4. pH of buffer/mobile phase
5. Composition of Mobile Phase
6. Selection of Column
7. Optimization of Analyte signal
8. Objectives of Seperation
8.1 Analysis time
8.2 Resolution
8.3 k factor (Capacity factor)
8.4 Peak height
8.5 Asyymetry
8.6 Theoritical Plates
9. Flow rate
14.viod volume and dead time

It is the volume of the mobile phase required from

reservior to detector

The time when the unretained solute elutes from the column
after the injection, is called dead time.
15.what is least count of a balance? How we find out this? Any formula for
least count of Analytical balance?

A minimum weight you can measured is called least count of

the analytical balance.

16.how to pepare 6.8 pH phosphate buffer

Add 6.8 gm KH2PO4 and about 0.94 gm NaOH in 1000mL Water.

It directly shows pH 6.8.
Adjust the pH of solution by using dilute Orthophosphoric
acid or NaOH.

6.8gm KH2PO4 + 0.94gm NaOH

17.Why the same type of columns showing different RT for a single

product?

because , when manufacturing of column is done different

parameter is affected which is given below , due to this
each column is unique tho it is same type.

column parameter

column pressure
parical size
carbon density
dead voulume of the column
end fitting
end capping of the column.
silica used.

Hplc System Parameter

length of tubing.
internal dia. of tube.
void volume of system.
pump working.
flow rate.
end fittings.
ETC....
18.what are the important specifications for a Column ?

Separation efficiency, Inertness, durability, Ph range and

batch to batch reproducibility.
19.Why we use holmium oxide,perchloric acid for uv/vis calibration

IN THE PARAMETER OF CONTROL OF WAVELENTH

ONLY HOLMIUM OXIDE CAN GIVE ALL PEAKS
AT PERTICULAR WAVELENTH IN BETWN 200 NM TO 600 NM.
20.1 % solution is equal to how much ppm

1 ppm= 1 mg/L

1% is
= 1 g in 100ml
= 1000 mg in 0.1 L
= 10000 mg/L
= 10000 ppm
21.what r the reasons to get peak telling?

Due to column efficiency Decreaced

22.what is the difference between M.C and LOD

M.C =moisture content= the molecular water stored in the

molecule/it gives the exact water centent.(generally tested
by the KF titrator)

whereas

LOD=Loss on drying= unbound water + organic volatile

impurities.(in vacuum oven)
23.what is regration method in method validation , how we can calculate
it.

Regression method is showing your method capability over

the specification range and it should be 0.99
24.What is the composition of K.F

It is mixture of imidazole or any primary amine,SO2,I2

containing methanol or 2-(2-Ethoxyethoxy) ethanol as
diluent.
25.what is peak purity ??

IT IS DEFINED AS PURITY OF A PERTICULAR PEAK THAT IT IS NOT

COELUTED WITH OTHER IMPURITIES

PURITY=RATIO OF ABSORBANCE B/W THE ROOT OF UP SLOPE AND

APEX/RATIO OF ABSORBANCE B/W THE ROOT OF DOWN SLOPE AND APEX

IF THE PURITY=1, PURE PEAK

26.How are PDA Detector work in HPLC

PDA MEANS PHOTO DIODE ARRAY DETECTOR WHICH IS USED TO FIND

THE RETENTION TIME OF THE PEAK AT MULTIPLE WAVELENTH i.e at
DIFFERENT NANOMETERS IN HPLC INSTRUMENT.FOR EXAMPLE CAFFINE
WAVELENTH CAN BE DETECT AT 205, 243, 273 nm AT A TIME BY
PDA DETECTOR BY HPLC
27.why we are using polystyrene film for calibration of I.R spectroscopy?

polystyrene film is made by the polymer of styrene.its

having highly durability and stabeled at any temparature
because its made by the polymers.
28.what difinition of Precision.

precision posseses different meanings but .a simple ay to

explain precission is that....

" its the meaure between the data obtained in any

experiment which is close to pairs of each other but that
data is ,, PRONE TO ERRORS AND UNACCURATE,,& NON-
REPRODUCIBLE"
29.IN DISSOLUTION TEST S1AND S2 AND S3 CONDITIONS ALL ARE FAILED
THEN WHAT IS THE DESSICION U CAN TAKE

Phase I investigation should be done .

As a part of Investigation,Check the previous trend data and development of
the product.Root cause is not identified then raise the OOS.
Second officer results also found OOS. We can conclude that the OOS results
are due to the product behavior.
30.Why sodium lamp is used as source of Polarimeter?

a. Gives high-energy output.

b. High sensitivity and S/N ratio.
c. Line spectrum i.e. gives monochromatic light of
589nm
31.What is Specificity ?

this parameter is used to check the purity's of sample and impurities which
we are used in validation,Retention time identification,check the separation
of impurities and also used to calculation of RRT AND RRf's
32. what is the difference between melting range & melting point? Which is
more significant?

melting range means range of temperature at which

temperature sample startsmelting(solidform to liquid form)
and at which temperature completely converted to liquid
form.example 121.4-123.6

melting point means sample melts sharply one point of

tmperature , example 82
32.Why 10%,20%,30% Sucrose solution is used for the calibration of
polarimeter?

for linearity purpose and calculation of correlation

coefficient

33.In HPLC what is Single point threshold, peak purity index and minimum
peak purity index? what should be consider for pure peak.

Single point threshold is show only peak maxima spectra

whenever peak purity index is shows the front, tail and
peak maxima spectra.

for pure peak we have to consider three point peak purity

which shows that your purity angle should be less than
purity threshold which is clearly shows that your peak is
specrally pure and homogeneous.
34.why there is a need of optical activity & specific optical activity
measurement by polarimeter

For chiral compounds,which are having steoreo isomers.

35. Why Methanol used in KF Titration?

During titration Py.SO3 can react with water which varies

the stoichiometry of water & iodine from 1:1 to 2:1.To
prevent such side reaction methanol is added

36.on what basis SOP's can be changed? IF mobile phase ratio is 70:30 to
how much % can we change the ratio in HPLC process

+- 10% as per usp guideline mp ratio can be changedrevised

SOP must be there for reason /justification inclusion SOP
Can be modified
37.is ther any guidline is available for limt of peak fronting.and what is
the limt of peak fronting.

Peak fronting or peak tailing is onr of the parameter of

peak shrpness.
It shuold be between 0.8 and 1.5 as per USP.

38.which solvent is highest poler acetonitrile dimethyl formamide

methanole tetrahydrofurane buffer solution

Non polar compounds having low dielectric

constant .according to the above list

acetonitrile = 36.6

DMF = 38.3

METHANOL =33

THF =7.52

Based on the above dielectric constant , DMF is the

polar solvent.
39.what does optical rotation and specific rotataion means,difference
between them

when sample place in polarimeter and the reading shows how

much plain polarized light is rotated is called optical
rotation

&

Specific Optical Rotation=(100*Optical Rotation)/(Cell

Length in Decimeters*Concentration)
Here 1mm = 0.01 Decimeter
40.How will you separate Colorless compound Using Column
Chromatography?

in colourless compond seperatiion each 100ml fraction

collected seperately and using Thinlayer chromatography
check the seperated fraction by using UV lamp
41.Why do we use 0.005M h2so4 solution for preparation of k2cr2o7
solution for UV calibration?

we use 0.005M of sulphuric acid because pottasium

dichromate is not easily soluble in distilled water,so
dissolve K2cr2o7 we use ulphuric acid
42.why we cannot analysis below 200nm in u.v?

In UV region below 200nm is a vaccum UV where there is a

characteristic UV absorptions taking place by atmospheric
Oxygen. Thats why we dont perform analysis in Vaccum UV
egion
43.How to choose Normal phase or Reverse Phase HPLC method
Conditions for given sample?

based on sample nature i.e if it polar in nature we can

choose reverse phase if it is non polar in nature we can
choose normal phase

44.HPLC Calibration was done in Reverse Phase, but not in normal phase
why explain?

Reverse phase and Normal phase classification is based on

the mobile phase and stationary phase (column).But we're
calibrating the HPLC for PUMP,COLUMN OVEN,AUTOINJECTOR and
DETECTOR etc.So we can perform the calibration for HPLC
with Normal phase also.But the ease availability of column
and mobile phase of Reversed phase, it is widely use for
the HPLC calibration.
45.is there any material other than caffene to calibrate hplc detector

naphthalene,benzophenone, caffene as per pharmacopeias

46. What is the formula for Tapdensity

Tapped Density- W/V

where W is weight of sample
V is final tapped volume
this is as per USP
47.what is a drug?

Drug is a any chemical substance used for

prevention ,meetigation,cure or treatment of any disease or
disorder in living organisms ,when administered into the
body absorb in the blood and gives the therapuetic action.

48.how can standardized of HCl of 0.1 N

By using 100 mg of sodium carbonate in 100ml of water,methyl

orange as an indicator.

Normality of 0.1N HCl=wt of Na2Co3*0.01/Vol.of 0.1N HCl*.0053

49. what is the value of pH
pH is the minus logarithum of the concentration of H+ ion to
the base 10..scale 0 to 14 (discovered by SORENSON)
Less than 7-Acidic in nature
Greater than 7-Basic in nature
and equal to 7-neutral in nature
1st crireria is the soln must be an aqueous soln..
2nd thing its must be an electrolyte..
we can see pH value only that soln whose concentration is
below or equal to 1M...
50.what is protocol ? plz Explain me

Its contains all information of your product.Likestability

or any one at which condition sample kept at temperature,
tests required, sample quanity, time period,
specifications, raw material sorces, pacaking materials
etc...
51.a bottle of concerntrated HCL (36.5) was labelled as 37% (w/w) and
density 1.19m/l.What is hcl molarity?

the molarity of the resultant solution will be around 12

molar
molarity=1.19*1000*37/100*36.5=12 m
52.why kf factor coming 0.5

The question is wrong. It should be "why kf factor coming

0.5mg/ml".

The answer is because 0.5mg of water is neutralised by one

ml of kf reagent.
53.what is the lamp used in SOR?

Sodium vapour lamp produces two distinct wavelengths D1 and

D2 lines respectively one at 589nm and 589.6nm if u observe
closely averaging out to 589.3nm.

Mercury Lamp is also used for a wide range of wavelengths

since it is a white light.
54. what is relationship between absorption and transmittance in u.v.?

A = (1-Log T)

(Absorbace is equal to one minus log of Transmittance to

the base 10)