Glassware SOP

1.
SCOPE AND LIMITATION OF METHOD:

The objective of this calibration procedure is to verify the error in Volume Glassware /Measure as
per National & International Standards i.e. For Piston pipettes range 10 to 10000 µl, ISO 8655-6
is referred and for Glass pipettes above 1 to 1000 ml and other glassware ISO 4787:2010 is
referred.
2. REFERENCE STANDARD / EQUIPMENT:
Sr. Equipment Resolution Range

No.
1. Analytical weighing balance 0.01/0.1 mg 82/220 g
2. Weighing balance 0.01 g 3100 g
3. Glass Thermometer for water 0.10C - 40 to 600C
temperature measurement
4. Barometer for Monitoring of Air 0.1 0C, 1 %, 0.1 hPa 0 to 50 0C, 10 - 90 % ,
Pressure, Air Temperature & 10 to 1100 hPa
Humidity
3. TERMS & DEFINITIONS:

3.1 Fixed volume Pipettes -
Those which are designed and supplied by the manufacturer to dispense a specific, fixed
volume of liquid (defined as the nominal volume). This volume cannot normally be altered,
although some types are designed so that they can be adjusted within small limits by the user
to compensate for errors found during calibration or for use with liquids having physical
properties differing from water.
3.2 Variable volume Pipettes -

Those in which the user can adjust the volume of liquid to be dispensed over a range specified by
the manufacturer. In this case the nominal volume is defined as the upper limit of the
manufacturer’s designated volume range.
3.3 Maximum Permissible Error –

Upper & Lower permitted extreme value for the deviation of the dispensed volume from the
nominal volume or selected volume of the volumetric instrument.
4. PRINCIPLE OF CALIBRATION:
The basic principal of gravimetric method is to weigh the amount of distilled water delivered in a
single operation of the volumetric instrument, thus giving volume of the water. A number of repeat
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measurements are made to which corrections must be applied to compensate for any variation
from standard temperature and atmospheric conditions, and any significant evaporation of the
water during the test period.
For precision measurement of capacity of volumetric Glassware /Measure, the gravimetric
method is used.
In this method capacity is determined by weighing the quantity of distilled water which the
measure contains or delivers at the temperature of the liquid & applying a correction to convert
the mass of water to volume at the reference temperature.
5. PREPARATION:
5.1 Preparation of piston pipette under calibration:
a. Check the identification.

b. Ensure smooth working of pipette and visually examine any damages of pipette condition.
c. Ensure pipette put in lab on worktable for 1 hr to reach equilibrium with the room
conditions.
d. Test water is kept in the lab for 1 hr, to reach equilibrium with the room conditions.
e. Ensure lab environmental conditions e.g. temperature, humidity, air pressure meets as per
specifications.
5.2 Cleaning & Handling of Glassware:
The volume contained in, or delivered by, a volumetric instrument depends on the cleanliness of
the internal glass surface. Lack of cleanliness results in errors through a poorly shaped meniscus
involving two defects:
 - incomplete wetting of the glass surface, i.e. the liquid surface meets the glass at an arbitrary
angle instead of forming a curve such that it meets the glass tangentially;
- a generally increased radius of curvature, due to contamination of the liquid surface reducing
the surface tension.
a) General laboratory practice is to cleaning rinsing and drying of lab glassware
Cleaning:
Clean all glassware before use as a general practice. Use chromic acid or aceton to clean the
glassware to clean the glassware. Avoid brushes and cleaning pads which could abrade the
glass or damage the coating. If using a chromic acid cleaning solution minimize contact of the
solution with coating.
Glassware must be Absolutely Grease-Free. The safest criteria of cleanliness are uniform
wetting of the surface by distilled water. This is especially important in glassware used for
measuring the volume of liquids. Grease and other contaminating materials will prevent the
glass from becoming uniformly wetted. This in turn will alter the volume of residue adhering to
the walls of the glass container and thus affect the volume of liquid delivered. Furthermore, in

pipettes and burettes, the meniscus will be distorted and the correct adjustments cannot be
made. The presence of small amounts of impurities may also alter the meniscus.
For Handling & Storing
To Prevent Breakage of glassware like pipettes, cylinder, or burettes, be careful not to let tips
hit the sink or the water tap. Protect clean glassware from dust. This is done best by plugging
them with cotton, corking, or placing the glassware in a dust-free cabinet.
5.3 Preparation of glassware under calibration:

a. Check the identification, date of receipt of item.
b. Clean & dry the glassware to be calibrated, before calibration.
c. Ensure UUC, put in lab on worktable for 1 hr to reach equilibrium with the room
conditions.
d. Distilled water is kept in the lab for 1 hr, to reach equilibrium with the room conditions.
e. Ensure environmental conditions and Record environmental conditions and item
descriptions in the relevant calibration data sheet. e.g. temperature, humidity, air pressure
meets as per specifications.
f. The meniscus can be highlighted by holding a white piece of paper with a heavy black mark
on it behind the glassware.
5.4 Preparation of reference equipment & apparatus:

a. Clean the worktable, pan, digital display of analytical balance.
b. Ensure smooth operation with zero setting and proper leveling of analytical balance.
c. Ensure analytical balance on worktable for 1 hr to reach equilibrium with the room
conditions.
d. Ensure proper working of digital barometer for monitoring of temp., humidity and air
pressure.
e. Deliver the pipettes & tips into lab at least 1 hr before the calibration
f. Prepare the working area: Tips in racks, weighing vessel, distilled water. Balance &
surrounding surface table is cleaned from dust & other impurities.
5.5 Preparation of test distilled water:
At the start and the end of the weighing procedure, the temperature of the test water will
to the nearest 0.20C
5.6 Preparation of test distilled water:

a) Room temperature 210C to 270C

(The temperature should be stable to within ± 0.50C)
b) Relative Humidity 50 ± 10% RH
6. CALIBRATION METHOD :
6.1 DETAILED CALIBRATION METHOD FOR PISTON PIPETTE (As per ISO 8655-6):
1. Ensure water in the balance water container at least half full. If the water level is too low, add
distilled water. Do not overfill the container. Ensure water in the pipetting container to a depth at
least 3 mm.
2. Press set zero key and ensure set zero of balance display.
2. Fit the suitable tip to the pipette.
3. Fill the tip with distilled water and expel to waste five times to reach humidity equilibrium in the
dead air volume of the pipette.
4. Calibrate the pipette at least three volumes i.e. nominal volume, 50% of the nominal volume,
10% of the nominal volume & For fixed volume pipette at nominal volume.
6. Set a range on pipette with rotating the cap clockwise or anticlockwise direction. Hold the
pipette in hand vertically and follows the process following ways shown in fig.1,
7. Press the operating button to the first stop. immersing the tip 2 to 3 mm below the surface of
distilled water
8. Release the operating button slowly to the ready position. This action will fill the tip with the
distilled water. Remove the tip vertically and carefully from the surface of distilled water.
9. Open the top sliding cover of analytical balance. Take the tip near to weighing vessel vertically.
Press the operating button to the second stop, and completely empty the tip in weighing vessel.
10. Release the operating button to the ready position. Remove the tip vertically and carefully from
the top sliding cover of analytical balance and close the top sliding cover.
11. Repeat the above same procedure for 10 measurements at each pre-decided volume and
record these readings in calibration data sheet.
Calculate measured volume using following formula :

V = (m2-m1) x (ρw – ρa )-1 x (1- (ρa / ρb) x [1- ϒ (tw – 27)]

Where,
m2 is the balance reading of vessel with water, in grams;
m1 is the balance reading of empty vessel, in grams (zero in case the balance was tared with the
volumetric instrument or receiving vessel);
ρw is the density of water at tw °C, in gm /ml
ρa is the density of air, in grams per millilitre, obtained from equation below at the
temperature and atmospheric pressure of the test;
Pa is the pressure, expressed in hectopascals (hPa);
Ø is the relative humidity, expressed as a percentage;
ta is the air temperature, expressed in degrees Celsius;
12. Affix the calibration sticker indicating its identification and calibration date at appropriate place
on the instrument and prepare the calibration Certificate having contents as per 5.10 of Quality
Manual.
6.2 DETAILED CALIBRATION METHOD FOR GLASSWARE (As per ISO 4787:2010):
Filling and Delivery of Volumetric flasks and measuring cylinders:

1 Glassware, which is to be calibrate, is cleaned and dried after cleaning.
2 Place the empty glassware, which to be calibrate, and tare the. The balance reading (m1) is
noted in the calibration data sheet.
3 Fill the glassware with distilled water using plastic tube with tip to a distance of few millimeters
above ring mark or graduation line to be calibrated, so that the walls of the volumetric
instrument considerably above the ring mark are not wetted.
4 The final setting of the meniscus to the ring mark or graduation line is made by withdrawing the
surplus water by means of a plastic tube drawn out to a jet. The movement of the meniscus
when setting is set to downwards.
5 Fill the glassware with distilled water up to the mark. Then dry up the outer surface of the
glassware with blotting paper.
6 Note down the temperature (tw) of the water, with a glass thermometer.
7 Place the glassware full of water on the pan of the balance, balance reading (m2) is noted in the
calibration data sheet.
8 Note down the air temperature, air pressure and relative humidity of air of the room, in the
9 Find the difference of the reading of the empty vessel & the filled vessels. (m=m2-m1).

Where,

ρa is the density of air, in grams per millilitre, obtained from equation below at the temperature and
atmospheric pressure of the test;
Filling and Delivery of Pipettes:

1 Pipettes is clamped in a vertical position and filled through the jet to a few millimetres above the
graduation line to be calibrated; any liquid remaining on the outside of the jet is removed.
2 Place the empty receiving vessel, which to be calibrate, and tare the. The balance reading (m1) is
3 The final setting of the meniscus is made by running out the surplus water through the jet. Any
drop of liquid adhering to the jet is removed, for example by bringing a ground glass surface
into contact with the tip of the jet at an angle of about 30°.
4 Draw this ground glass surface downwards through a distance of about 10 mm to remove
residual water. Delivery into the tared receiving vessel is made with the flow unrestricted while
the tip of the jet is in contact with the inner ground surface of the receiving vessel, finally
drawing it over a distance of about 10 mm, with the receiving vessel held inclined at an angle of
about 30°.
4 Determine the delivery time while the tip of the jet is in contact with the inner surface of the
receiving vessel, above the level of any collected liquid, but without movement of one against
the other throughout the delivery period.
8 Place the receiving vessel with water on the pan of the balance, balance reading (m2) is noted
in the calibration data sheet.

V = (m) x (ρw – ρa )-1 x (1- (ρa / ρb) x [1- ϒ (tw – 27)]
Where,

Filling and Delivery of Burette:
1 Burette is clamped in a vertical position and filled through the jet to a few millimetres above the
graduation line to be calibrated.
2 Place the empty receiving vessel, which to be calibrate, and tare the. The balance reading (m1) is
3 The stopcock and jet is freed from air bubbles. Any liquid remaining on the outside of the jet is
removed. The final setting of the meniscus is made by running out the surplus water through the
jet.
4 Any drop of liquid adhering to the jet is removed by bringing a ground glass surface into contact
with the tip of the jet at an angle of about 30°. Draw this ground glass surface downwards through
a distance of about 10 mm.
7 Delivery into the empty receiving vessel is made with the flow unrestricted until the meniscus has
come to a few millimetres above the graduation line to be calibrated, while the stopcock is fully
open and the jet is not in contact with the receiving vessel. After the final setting of the meniscus,
any drop of liquid adhering to the jet is removed by bringing an inclined glass surface into contact
with the tip of the jet at an angle of about 30°, finally drawing it over a distance of about 10 mm.
8 Place the receiving vessel with water on the pan of the balance, balance reading (m2) is noted in
the calibration data sheet.

Where,
7. General calculation:
Where,

ρw is the density of water at tw °C, in grams per millilitre, calculated with the equation below –
(a0)+((a1*10^(-2))*L26)+((a2*10^(-3))*(L26^2))+((a3*10^(-5))*(L26^3))+((a4*10^(-7))*(L26^4))
Where,
with the constants (ITS-90 temperature scale):
a0 is equal to 999.85308 kg/m3;
a1 is equal to 6.32693x10–2 °C–1 kg/m3;
a4 is equal to 3.821216x10–7 °C–4 kg/m3.
Determination of water Density (pw)

The density of water ρw (in kg/m3) is given by : (formula as per ISO/TR 20461)
 w  (999.85308) + ((6.32693*10^ (-2))* t w + ((-8.52382910^ (-3))* t w ^2)) + ((6.943248*10^ (-5)) * t w ^3)) + ((-3.821216 *10^ (-7))* t w ^4))
Where
t w = water temperature in degree Celsius;
Determination of Air Density (pa)

The density of air ρa (in kg/m3) is given by : (formula as per OIML R-111-1: 2004)
0.34848 p  0.009h  exp(0.061 Ta )

a  ( )
273.15  Ta
Where
p = pressure, expressed in hecto-pascals (hPa);
h = relative humidity, expressed as a percentage;
Ta = air temperature, expressed in degrees Celsius;
ρb is either the actual density of the balance weights when these are adjusted to their
nominal mass, or the reference density for which the weights have been adjusted (see
the note below), in grams per millilitre, or, when using an electronic balance without
weights, the (reference) density of the weights with which it has been adjusted; (density
of the weights is taken as 8 g/ml),
ϒ is the coefficient of cubical thermal expansion of the material of which the volumetric
instrument tested is made, in reciprocal degrees Celsius,

where, Coefficient of cubical Coefficient of cubical
for Borosilicate glass 3.3 = 0.0000099 /0C,
for Borosilicate glass 5.0 = 0.0000015 /0C,
for Soda-lime glass= 0.0000027 /0C,
for Piston Pipette = 0.000045 /0C.
tw is the temperature of the water used in testing, in degrees Celsius.
Volume at ta°C (V)=

V=[(m2-m1)/( ρw – ρa)] x (1- ρa / ρb) x (1- ϒ (tw – 27)
6. Uncertainty of Measurements
Uncertainty evaluation in calibration of Volume of Glassware / Measure
TYPE A
Standard uncertainty due to Repeatability of readings is Type A uncertainty (UA).

Type A evaluation is associated with normal probability distribution. If X 1, X2, X3, Xn are the observations
made during measurement, then standard deviation to be calculate as follows:
σ = √{ 1/ n-1 Σ (Xi – X) 2 }
Now, Standard deviation of mean or the standard uncertainty for type A is given by,
UA = σ / √n
Where,
σ is Standard deviation,
n is number of Observation &
UA Standard deviation of mean or the standard uncertainty.
Degrees of freedom
νi = n – 1
TYPE B
a) Type B components For Micro-Pipette (As per ISO/TR 20461)
(i) U1 = Uncertainty due to Uncertainty of standard (Weighing Balance) – from calibration certificate.
(ii) U2 = Uncertainty due to Repeatability of weighing balance used for calibration,
(iii) U3 = Uncertainty due to water temperature (considering accuracy and uncertainty of glass
thermometer used for water temperature measurement),
(iv) U4 = Uncertainty due to air temperature (considering accuracy and uncertainty of thermo-
barometer used for air temperature measurement),
(v) U5 = Uncertainty due to air pressure (considering accuracy and uncertainty of thermo-barometer
thermometer used for air pressure measurement),

(vi) U6 = Uncertainty due to humidity (considering accuracy and uncertainty of thermo- barometer
used for humidity measurement),
Sensitivity coefficients are calculated using following formula (as per ISO/TR 20461):
i) Sensitivity Coefficient for Water Temperature :
4
m
Ctw     iatwi 1
w2 i 1
ii) Sensitivity Coefficient for Air Temperature :
m  (k 2ta 0  k 3)  k1 pa
Cta  
w 2 (ta  ta 0) 2
iii) Sensitivity Coefficient for Air Pressure :
m k1
Cpa  
w (ta  ta 0)
2
iv) Sensitivity Coefficient for Relative Air Humidity :

m k 2ta  k 3
C  
w2 (ta  ta 0)
b) Type B components For Glassware
(i) U1 = Uncertainty due to Uncertainty of standard (Weighing Balance) – from calibration certificate.
(ii) U2 = Uncertainty due readability of weighing balance
Combined standard uncertainty
uc =  ( uA2 + u12 + u32...+ un2)
Effective degrees of freedom (veff)
νeff = (n-1) [(uc)4/(uA)4 ]
Using value of νeff , Value of k is calculated from the following Table .
Table
Coverage factor k for various effective Degree of Freedom with coverage probability of 95 %
νeff 1 2 3 4 5 6 7 8 9 10 11 12
k 12.71 4.30 3.18 2.78 2.57 2.45 2.36 2.31 2.26 2.23 2.20 2.18
νeff 13 14 15 17 20 25 30 ∞
k 2.16 2.14 2.13 2.11 2.09 2.06 2.04 1.96

Expanded Uncertainty
U = k x uc
7. REF IS STANDARD:
TYPE OF CALIBRATED ITEM STANDARD VALUE

Laboratory Glassware-Graduated Measuring Cylinders IS 878:2008 (ISO 4788:2005)
Laboratory Glassware-Burettes IS 1997:2008 (ISO 385:2005)
Laboratory Glassware-One-Mark Volumetric Flasks IS 915:2012 (ISO 1042:1998)
Laboratory Glassware-Graduated Pipettes IS/ISO 835:2007(Reaffirmed-2012)
Indian Standard Specification for one-mark pipettes IS:1117-1975 (Reaffirmed-2012)
Laboratory glassware-Volumetric Instruments-Methods for
ISO 4787:2010
Testing of capacity and for use
Piston-operated volumetric apparatus -- Part 6:
ISO 8655-6:2002
Gravimetric methods for the determination of
measurement error
Determination of uncertainty for volume
measurements made using the gravimetric ISO/TR 20461
method
8. RECORD REF:
Calibration Data of Volume – IF-40


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1.

SCOPE AND LIMITATION OF METHOD:

2. REFERENCE STANDARD / EQUIPMENT:

Sr. Equipment Resolution Range

3. TERMS & DEFINITIONS:

3.2 Variable volume Pipettes -

3.3 Maximum Permissible Error –

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5.1 Preparation of piston pipette under calibration:

a. Check the identification.

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5.3 Preparation of glassware under calibration:

5.4 Preparation of reference equipment & apparatus:

5.6 Preparation of test distilled water:

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Calculate measured volume using following formula :

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Filling and Delivery of Volumetric flasks and measuring cylinders:

Calculate measured volume using following formula :

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Filling and Delivery of Pipettes:

Calculate measured volume using following formula :

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Calculate measured volume using following formula :

V = (m2-m1) x (ρw – ρa )-1 x (1- (ρa / ρb) x [1- ϒ (tw – 27)]

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Determination of water Density (pw)

Determination of Air Density (pa)

0.34848 p  0.009h  exp(0.061 Ta )

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Volume at ta°C (V)=

Uncertainty evaluation in calibration of Volume of Glassware / Measure

Standard uncertainty due to Repeatability of readings is Type A uncertainty (UA).

a) Type B components For Micro-Pipette (As per ISO/TR 20461)

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ii) Sensitivity Coefficient for Air Temperature :

iv) Sensitivity Coefficient for Relative Air Humidity :

Combined standard uncertainty

uc =  ( uA2 + u12 + u32...+ un2)

Effective degrees of freedom (veff)

νeff = (n-1) [(uc)4/(uA)4 ]

Using value of νeff , Value of k is calculated from the following Table .

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TYPE OF CALIBRATED ITEM STANDARD VALUE

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