Analysis of moisture is becoming increasingly important to many different industries Many of these reasons require not only a precise, but also accurate reading For years, most moisture analyses have been conducted on a semi-quantitative or even qualitative level

1. Handling Difficult Samples in
Karl Fischer Analysis

2. Background
• Analysis of moisture is becoming
increasingly important to many different
industries
• Many of these reasons require not only a
precise, but also accurate reading
• For years, most moisture analyses have
been conducted on a semi-quantitative
or even qualitative level

3. Questions about how much water?
Karl Fischer analysis is the answer.
• Karl Fischer (KF) titrations have been the gold standard for
water analysis since 1935
• The KF titration provides a reliable and robust way to directly
analyze for water in a sample
• Many other techniques exist for indirect analysis of water in a
sample, but KF titration is the best way to calibrate this other
methodology
• KF is rapid, well characterized, and the recognized standard
for water analysis in many solids, liquids and semi-solids
CH3OH + SO2 + RN → (RNH)SO3CH3
H2O + I2 + (RNH)SO3CH3 + 2 RN → (RNH)SO4CH3 + 2 (RNH)I

5. Measurement range
 % range  ppm range
Volumetric KF titration Coulometric KF titration

6. Volumetric KF titration
Direct addition of reagents by burette to sample
in methanol

7. Typical volumetric KF titrator

8. Coulometric KF titration
In situ generation of iodine by electrical means

9. Typical coulometric KF titrator

11. Most KF systems I see can only
handle one sample at a time!
Coulometric KF
Volumetric KF

12. Karl Fischer reaction
CH3OH + SO2 + RN (RNH)SO3CH3
H2O + I2 + (RNH)SO3CH3 + 2 RN (RNH)SO4CH3 + 2 (RNH)I
Hydroxide,
CarbonateReducing agent
Oxidant
Silanol
Acid Acid / Base
Ketone /
Aldehyde Aldehyde

13. Side reactions
• Acids
• Bases
• Aldehydes and ketones
• Carbonates, hydroxydes and oxides
• Silanol / Siloxane
• Strong reducing agents
• Strong oxidizing agents

14. KF titration curve
Fast release of water
Slow release of water
~130 s ~290 s

16. How do we mitigate side reactions,
poor water release and gaseous
samples?
• Adjust pH with buffers
• Use specialized reagents for aldehydes
and ketones
• Use different sample preparation

17. What if my sample comes in a
container that looks like this?

18. KF Gas Analyzer
• For determining water in:
• Propane
• Propene
• LPG
• LNG
• Butane
• Butene
• Butadiene
• Dimethyl ether
• Ethylene oxide
• Methylene chloride
• Ethylene chloride
• Vinyl chloride
• Chlorofluorocarbons
• Analyzes gases and liquefied
gases from sample cylinders!

19. Mass Flow
↙ Controller
← Oil Filter
Heater →
Precision
← Control
Valve
↙ Coulometer
Sample
Inlet→
Nitrogen
Inlet→
KF Gas Analyzer

20. Reproducibility
Results of 360 measurements of LPG (3 g per measurement) within 84 h, mean value: 26,5
µg/g ± 0,8 µg/g
y = -0,005x + 27,389
0
5
10
15
20
25
30
35
0 50 100 150 200 250 300 350
number of measurements
watercontent[µg/g]
80°C / constant sample flow
50°C / constant
sample flow
50°C / fluctuating sample flow

21. What if my sample is solid or liquid
and needs a lot of preparation?

23. Gas extraction
1. Samples
soluble in KF
reagent
2. Samples that
release water
in KF
compatible
solubility
promoters
direct titration gas extraction
1. Samples that
are not
soluble in KF
reagent
2. Samples that
cause side
reactions
liquid extraction
1. Samples that
release water
in organic
liquids

24. Principle of the Metrohm oven
technique
Dry carrier gas is used to
transport evaporated humidity
into titration vessel
Universal to nearly all samples

25. Principle of the Metrohm oven
technique
Metrohm developed the orginal
headspace analysis for Karl Fischer
titration

26. Advantages of using a KF Oven
• Nearly all KF analyses can utilize oven
• Very economical
• Much smaller reagent use
• Much more effective use of time
• Efficient
• Automation to handle many samples without human
interaction
• No need to clean vessel after just a few analyses
• Wide variety of applications
• Nearly all KF samples can be adapted to oven
technique

27. Gas extraction (oven method)
• Step 1: (use empty vial)
- Heat up KF oven
- Start gas flow
- Titrate cell to dryness
• Step 2: (use empty vial)
- Determine blank value
• Step 3:
- Put sample vial into the oven
- Heat up sample and transfer released water with carrier
gas to KF cell

28. Gas extraction (oven method)
Sample: Motor oil
• Oven, 160°C, N2
Content: ~ 390 ppm
• Direct measurement
Content: ~ 960 ppm
long determination time
curve with side reaction

29. Headspace KF analysis
• Wonderful technique for most sample types
• Until recently, Metrohm sample changers would
only accommodate a 6 mL headspace vial
• Now Metrohm can adapt our 874 USB KF Oven
Sample Processor to fit many of the most common
vials

30. How do you adapt a sample to KF
Ovens?
• Choose a temperature
• Classic method
• Multiple samples with different temperatures
• Choose best temperature for most stable and
reproducible moisture evolution
• Optimize for speed
• Gradient Method
• One sample over a range of temperatures
• Choose a carrier gas
• Choose air, nitrogen or argon depending on the
thermal stability and combustibility of the
sample

31. Example data: method development
Classic method of temperature determination
• Steroidal compound
Temp. Air source Extraction Time Water
Content
Decomposition
?
1 150 Air 300 0.04% Yes
2 160 Air 300 4.44% Yes
3 170 Air 300 6.60% Yes
4 200 Air 300 6.91% Yes
5 150 N2 300 0.14% Yes
6 155 N2 300 0.98% Yes
7 135 N2 1800 0.04% No
8 140 N2 900 0.06% No

32. Temperature gradient
• What oven temperature is optimal for an unknown
sample?
• Find the ideal temperature running a temperature
gradient with the KF OVen
• Looking for a temperature that will accelerate
water extraction but not pyrolyze the sample
• Pyrolysis or combustion leads to erroneously high results

33. Example data from temperature
gradient analysis
• Sample of mixed sugars and buffers
• Lyophilized Sample
• Ran 50 – 180ºC in 30 minutes

34. Example data from temperature
gradient analysis
• Chose temperature of 95 degrees
• Analysis was slightly more than 7
minutes
• This only took 1 gradient sample and one
sample to confirm
• Without gradient, this takes up to 7
samples

35. Example data: method development
Gas determination
• Steroidal compound
Air carrier gas at high
temperature
Evidence of
decomposition
Nitrogen carrier gas at
low temperature
Stable extraction

36. Headspace Karl Fischer moisture
analysis in your vial
• The 874 uses Metrohm’s
exclusive KF Oven
technology for unsurpassed
moisture results
• The 874 is perfect for your
lyophilized samples because
Metrohm USA will customize
the 874 to fit your sample
vial!
• Because only water vapor is
analyzed, reagent costs are
greatly reduced
• Holds up to 36 samples
depending on rack
configuration

37. Metrohm NIRSystems
Positive vibration
Reagent-free Moisture Analysis
Dr. Hari Narayanan
Product Manager NIRS
Metrohm USA

38. Techniques for moisture analysis

39. Metrohm expands its portfolio to include
Near Infrared Spectroscopy!
<<Green Chemistry>>
Designed by FOSS
Powered by Metrohm

40. Techniques for moisture analysis

41. Why NIR for moisture analysis
• Requires no sample preparation
• Non-destructive
• Fast, accurate and precise
• Multi component analysis
• No solvent procurement or disposal
• Low-cost analysis
• Easy to use and maintain
• Remote sampling via fiber optics
Secondary technique: requires primary
method for calibration development

42. • Stretching
• Bending
symmetrical asymmetrical
scissoring rocking wagging twisting
NIR Spectroscopy
• 780nm to 2500nm
• Overtones and Combination Bands

43. • Based on absorption
in the Near Infrared
region of the
spectrum
• It provides both
chemical and
physical information
• Primarily sensitive to
organic compounds
• O-H, C-H, N-H bonds
absorb strongly in
NIR
NIR spectroscopy

44. NIR spectrum
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
700 900 1100 1300 1500 1700 1900 2100 2300 2500
wavelength (nm)
Absorbance(Log1/R)
Combination
1st Overtone
2nd Overtone
3rd Overtone

45. NIR spectrum of water
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
1100 1300 1500 1700 1900 2100 2300 2500
Wavelength (nm)
Absorbance(Log1/T)
Transmission spectrum of water
Pathlength ~ 3 mm

46. Sampling in NIR
Samples Types
Reflectance: Solids, Granules, Powders…
Trans-Reflectance: Pastes, Syrups…
Transmission: Liquids, Films, Oils…
Diffuse Transmission: Tablets, Softgels…
Analysis Types
• Identification
• Qualification
• Quantification
• Trend analysis – Determination of Change
• Process monitoring and Control

47. Identification & Qualification
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
1100 1300 1500 1700 1900 2100 2300 2500
Wavelength (nm)
Absorbance(Log1/R)
anhydrous lactose
lactose monohydrate
Water Bands

48. Run Samples as
Standards in NIR
Analyze
Samples by
Primary
Method
Calibration
and
Validation
Routine
Analysis by
NIR
Right Sampling Module
Sample Presentation
Collection Parameter
Accuracy of
Primary method
influences NIR
Method
Chemometric
Software
Method
Maintenance
Quantitative Moisture analysis

49. Instrumentation Pre-dispersive
Post-Dispersive: Scanning and PDA

50. XDS Monochromator
Hot-swappable modules

51. Metrohm NIRSystems
Family
Lab
NIRS XDS
Analyzer
NIRS XDS
RapidContent Analyzer
(RCA)
NIRS XDS
RapidContent Analyzer
– Solids
NIRS XDS
MultiVial
Analyzer
NIRS XDS MasterLab
Analyzer
NIRS XDS SmartProbe
Analyzer
NIRS XDS Interactance
OptiProbe Analyzer
NIRS XDS
Transmission
OptiProbe Analyzer
NIRS XDS RapidLiquid
Analyzer (RLA)
NIRS DS2500
Analyzer
Process
NIRS Analyzer PRO
NIRS Analyzer PRO
DirectLight/NonContact
NIRS Analyzer PRO
ContactReflection
NIRS Analyzer PRO
FiberSystem
NIRS XDS Process
Analyzer
NIRS XDS Process
Analyzer – SingleFiber
SinglePoint
NIRS XDS Process
Analyzer – SingleFiber
4 Channels
NIRS XDS Process
Analyzer – SingleFiber
9 Channels
NIRS XDS Process
Analyzer – Microbundle
SinglePoint
NIRS XDS Process
Analyzer – Microbundle
4 Channels
NIRS XDS Process
Analyzer – Microbundle
9 Channels
NIRS XDS Process
Analyzer –
DirectLight/NonContact

52. NIR Applications
• Pharmaceuticals
• Chemicals
• Petrochemicals
• Polymers
• Paper and Pulp
• Textiles
• Cosmetics
• And more…

53. Moisture in polymer pellets
The water content of expandable
polystyrene is determined
according to Karl Fischer
The sample is first dissolved in
p-xylene to extract the water
Then methanol is added to
precipitate the sample
The water is determined in the
supernatant solution by
coulometric titration

54. Moisture in polymer pellets
NIRS DS2500 Analyzer: Sample Rotation

55. Moisture in polymer pellets

56. Moisture in polymer pellets
• MLR
• R2 = 0.9988
• SEC = 0.5413
Lab Moisture %
NIRPredictedMoisture%
NIR Advantage
• Speed and Simplicity
• Reduce laboratory analysis
• Provide increased process
performance
• Quality assurance monitoring

57. Moisture in solvent and
recovery streams

58. NIRS XDS RapidLiquid Analyzer

59. Moisture in solvent mixture

60. Moisture in solvent mixture
PLS: Factor 3
Correlation: 0.9998
SEC: 0.1939
• Save Time & Money
• NIR can be off-line, at-
line, or on-line
• NIR can measure Solvent
components and moisture
in single run

61. Inline Process NIR
granulator drying analysis
• Single-pot
granulator dryer
• Probe in contact
with powder
• Provides real-time
process analysis
and control

62. Granulator drying analysis
• Blend of lactose and starch granulated with
1.5% water and 8% methanol
• Granulation was dried with heat and
vacuum
• NIR probe monitored drying in-process
• NIR prediction models developed for water
and methanol

63. NIRS XDS Process Analyzer

64. Granulator drying spectra
1800 1850 1900 1950 2000
-0.020
-0.016
-0.012
-0.008
-0.004
0.000
0.004
0.008
0.012

65. Granulator drying moisture analysis
Four factor PLS from 1800-2084nm
R2= 0.99; SEC=0.022; SECV=0.024

66. Granulator drying moisture trend
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
5:16:48 5:24:00 5:31:12 5:38:24 5:45:36 5:52:48 6:00:00 6:07:12 6:14:24 6:21:36
Moisture%
Time
Blend #4 Moisture % vs Time

67. Granulator drying methanol analysis
Four factor PLS :1800-2084nm;
R2=0.99; SEC=0.184; SECV=0.193

68. Granulator drying methanol trend
0
1
2
3
4
5
6
7
8
9
5:16:48 5:24:00 5:31:12 5:38:24 5:45:36 5:52:48 6:00:00 6:07:12
MeOH%
Time
Blend # 4 MeOH % vs Time

69. Moisture analysis in a fluid bed
dryer

70. Sampling
• Fluid bed dryer charged
with lactose and MCC
• Spectra collected every 50
seconds
• LOD sample taken every 5
minutes

71. View of spoon probe

72. Spectra of dryer samples

73. Prediction model
2 Factors PLS
1100 nm – 1650 nm,
SEC = 0.7358 %,
R2 = 0.9519.
Validation Example
SEP = 0.204 %

74. Trend chart of fluid bed
0
2
4
6
8
10
12
0 5 10 15 20 25 30 35 40 45
Time minutes
Moisture%

75. Moisture in lyophilized material
KF
Calibration
Validation
NIR
Routine Samples

76. NIR instrumentation
• NIRS XDS Rapid Content Analyzer
• Scan range
400–2500 nm
• 32 scans/spectrum

77. Experimental design
• Lyophilized product
• Contained in ~ 30 mL serum vials
• Average cake weight is 1.6 g
• Acceptable moisture limit is NMT 2 %
• Calibration sample set
• 46 samples used to prepare training and test sets
• Near-IR analysis
• Spectra of calibration samples collected in triplicate
• Primary analyses
• Regression equations developed using data from
volumetric Karl Fischer titration

78. Raw NIR absorbance spectra

79. Calibration for low residual
moisture
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
NIRPredicted(%moisture)
Karl Fischer (% moisture)
NIR Predicted % Moisture = 0.806 - 19.411
A"
A"
1844 nm
2124 nm






SEP = 0.119
R = 0.992

80. 3.95
1.00
0.74
1.19
0.95
1.09
1.09
9.52
1.30
1.38
3.6
0.7
0.8
1.0
0.8
1.1
0.8
9.0
1.1
1.3
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
9 89 144 183 203 241 266 270 287 290
%Moisture
Sample Number
NIR
LOD
Comparison of NIR and LOD
methods
LOD values were, on average, lower than NIR
results by 0.20 ± 0.17 % H2O.

81. Conclusion
• NIR is well suited for analysis of residual moisture in
lyophilized products
• Samples can be easily altered to provide calibration sets
with varying amounts of moisture
• Regression equations should be optimized for the
specific moisture range of interest
• The speed of the technique allows large sample sets to
be analyzed, enabling NIR to be used either as a
diagnostic or inspection tool
• Combined KF Thermoprep with NIR allows the quick
calibration and validation

82. Summary
• Combined Titration and NIR
• Calibration and Validation
• Routine application
• Accuracy and calibration stability
• Methods from lab to process
• Flexible and customized sampling accessories
• Rugged instrumentation
• Continual technical and application support

83. Thank You