HPLC calibration process and ranges

1. Dadasaheb Balpande college of
pharmacy, Besa, Nagpur.
Short seminar
Prepared by : Himanshu M. Bhogekar
B-Pharm IV Year

3. Mobile Phase
Degasser
Pump
Column Thermostat
Auto-sampler (Injector)
Detector
Control Module
HPLC Instrument
HPLC:
• Analytical
technique.
• Separate, Identify
and quantify
components in the
mixture.

4. Reservoir 1 Reservoir 2
Pump 1
Gradient
Controller
Pump 2
Mixing
Chamber
Solvent
Conditioning
Column Injector Pre-column
Analytical
Column
Detector
Recorder
Instrumentation of HPLC

5. • Technique to collect the documented data evidence.
• Process to provide a result for sample to accepted range.
Objectives of Qualification
• Mainly perform to check the accuracy.
• Measure uncertainty in the result and deviation.
• Reduces the failure rate of analysis.
• Helpful for the build-up the confidence of analytes.
• Time saving process.

6. Instrument module Parameter to be checked Typical acceptance range
Solvent delivery system
(Pump)
Flow rate accuracy Isocratic Gradient
Isocratic Gradient ±5.0% ±2.0%
Injector Volume precision RSD ≤ 1.0%
Carry-over ≤ 0.2%
Multi-wavelength
detector
Linearity 𝑟2
≥0.99900
Wavelength accuracy ±2nm
Drift According to Manufacturer

7. Requirements:
1. Beakers.
2. Volumetric flask.
3. Calibrated analytical balance.
4. Calibrated chronometer.
Mobile phase used: De-gassed water.
Flow rate: 0.5ml/min, 1ml/min,
1.5ml/min
Time: 1200sec (20min), 598sec (9.9min),
395sec (6.5min),
Procedure
1.
•Weigh the empty collecting vessel accurately and note the
reading
2.
•Set the flow rate properly say 0.5ml/min.
3.
•Allow water to pass and collect in collecting vessel.
4.
•Weigh the vessel again and note the reading.
5.
•Repeat the whole procedure for 3 times by changing time and
flow rate.

8. Sr.
no.
Flow rate
ml/min
Wt. of empty
volumetric flask
Wt. of volumetric
flask with water
Wt. of water
(W2-W1)
Time
(sec)
Flow rate of
pump
Acceptance
criteria
1. 0.5 14.50 24.49 9.99 1200 0.49 0.4802-0.4998
Observation table
𝑉 =
𝑚
𝜌
∴ 𝑉 =
9.99
1
∴ 𝑉 = 9.99
𝑚 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑡𝑖𝑚𝑒.
𝜌 − 𝐷𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 𝑎𝑡 𝑟𝑜𝑜𝑚 𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒
𝑓 =
𝑉
𝑡
∴ 𝑓 =
9.99
20
∴ 𝑓 = 0.49
𝑓 − measured flow rate
𝑡 − 𝐸𝑙𝑎𝑝𝑠𝑒𝑑 𝑡𝑖𝑚𝑒 (min)
𝑉 − 𝑣𝑜𝑙𝑢𝑚𝑒 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑖𝑛 𝑚𝑙
𝐷 = 100 ×
𝐹 − 𝑓
𝐹
∴ 𝐷 = 100 ×
0.5 − 0.49
0.5
∴ 𝐷 = 2%
𝐷 − 𝐷𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 (%)
𝐹 − 𝑁𝑜𝑚𝑖𝑛𝑎𝑙 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒
𝑓 − 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒

9. Flow Rate accuracy (gradient method)
Procedure
Requirements:
1. Detector – 256nm
Mobile phase A : Degassed water
Mobile phase B : Degassed water + 0.5%
acetone.
Flow rate : 1.0 ml/min
Time %mobile phase A (water) %mobile phase B
(water+0.5% acetone)
0.0 100 0
10 90 10
10.1 50 50
20 50 50
20.1 10 90
30 10 90
30.1 0 100
Ripple gradient composition: % of noise of the 50% line from gradient
chromatogram.
%𝑅 = 𝑁
ℎ50 × 100
%𝑅 = 𝑟𝑖𝑝𝑝𝑙𝑒
ℎ50 = ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 50% 𝑙𝑖𝑛𝑒
𝑁 = ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑛𝑜𝑖𝑠𝑒
Limit: ≤ 0.2%

10. Observation table
Sr.no. Name Height of AB Gradient accuracy (AB)
1. Height at 10% 108124 9.86
2. Height of 50% 551808 50.3
3. Height of 90% 994505 90.7
4. Height at 100% 1096183 100.0
%𝐻 =
ℎ
𝐻
× 100
∴ %𝐻 =
108124
1096183
× 100
∴ %𝐻 = 9.86
%𝐻 − 𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑐𝑜𝑚𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛
ℎ − ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑙𝑖𝑛𝑒
𝐻 − ℎ𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 100% 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝐵
𝑑 = 10 − 9.86
∴ 𝑑 = 0.14
𝑑 − deviation
Time %mobile phase A (water) %mobile phase B
(water+0.5% acetone)
0.0 100 0
10 90 10
10.1 50 50
20 50 50
20.1 10 90
30 10 90
30.1 0 100

11. Volume precision and carry-over
Solutions:
Solvent A: Degassed water : methanol (40:60)V/V
Reference solution (a) : dissolve 15.0mg methyl-
paraben and 15mg propyl-paraben in solvent A and
dilute to 100 ml with the same solvent.
Reference solution (b) : Dilute 1.0ml of reference
solution (a) to 10 ml with solvent A.
Requirements:
1. Column
2. Flow rate: 1.0 ml/min
3. Detection: 254 nm
4. Injection volume:
• (“Short pathway” flow-cell, for example 5 to
10 mm): 20µL
• (“long pathway” flow-cell, for e.g. 60 to
85mm): 2µL
Procedure
1 × Solvent A (blank injection 1)
6 × reference solution (b)
1 × reference solution (a)
1 × solvent A (blank injection 2)
1 × reference solution (b)
Limits:
Repeatability of peak area: The RSD of peak area of methyl-
paraben and propyl-paraben in chromatogram of six
consecutive injections of reference solution (b) should be ≤
1.0%.
Carry-over: Peak area of propyl-paraben obtain from blank
injection 2 and peak obtain from reference solution (b) not
exceed 2 %. Corresponds to 0.2%.

12. Multi-wavelength detector (linearity)
Stock solution: 10 mg caffeine + 20ml water (conc. 0.5mg/ml).
Solution Process Concentration
A 10ml + 100ml water 50µg/ml
B 1ml + 100ml water 0.5µg/ml
C 2ml + 200ml water 1µg/ml
D 1ml + 10ml water 5µg/ml
E 2ml + 20ml water 10µg/ml
F 5ml + 50ml water 25µg/ml
Requirements:
1. Column
2. Mobile phase – Acetonitrile: degassed water (15:85
V/V)
3. Oven temperature -- 40°C
4. Flow rate – 1.0ml/min. Run time : 6 min
5. Detection – 273 nm
Working Solution
Limit : 𝑟2≥0.99900
Procedure
2 × Blank
1× B
1× C
1× D
1× E
1× F
y = 53956x - 2423.2
R² = 0.9998
0
500000
1000000
1500000
2000000
2500000
3000000
0 10 20 30 40 50 60
AREA
CONCENTRATION (ΜG/ML)

13. Multi-wavelength detector (Accuracy)
Solution: Caffeine 25µg/ml used.
Inject Caffeine
solution.
Record the spectrum
by scanning from
202 – 208 (Maxima),
270 – 276 (Maxima)
242 -248 (Minima)
Limit : ±2nm