HPLC-HPTLC Comparison

This presentation lays out all the differences between the two analytical techniques i.e. High Performance Thin Layer Chromatography and High Performance Liquid Chromatography.

For more information regarding HPTLC, please visit www.anchrom.in or you can send us an email at [email protected]

Thank you
Anchrom

1. HPTLC
&
HPLC
1

2. HPLC & HPTLC
Complimentary
Liquid Chromatography
Techniques
2

3. LC & HPTLC
Part I
COMPARISON
OF
TECHNICAL
MERITS
3

4. Active Phases
to manipulate separation
LC HPTLC
Solid Solid
Liquid Liquid
Gas
4

5. What is a Chromatogram?
HPLC HPTLC
Machine only Machine +
5

6. Separation Medium
HPLC Column
C-18
HPTLC Plate, 20x10 cm
Silica Gel 60F 254
6

7. Separation Power
LC HPTLC
High
To
Ultra High
Upto20-25
fractions / sample
Medium
to
High
Upto10-12
fractions / sample
HPTLC has enough separation powerfor routine analysis
7

8. Selectivity
LC HPTLC
Reverse Phase Normal Phase
Partition
(weak)
Adsorption
(strong)
Poor Very good
8

9. Operating Pressure
LC HPTLC
Very high
to
Ultra high
Atmospheric
2000-15000 psi 14 psi
Delicate Rugged
Expert required Not required
9
Requires a pump Capillary force

10. Sample
Analysis in
Parallel
Only one sample
at a time
6 different
samples, 20 of
each = up to
6 x 20=120 at
a time
Sample E
Sample D
Sample C
Sample B
Sample A
Sample F
10
HPLC
HPTLC

11. Sensitivity
LC HPTLC
High
to
Ultra high (MS)
Moderate
to
Ultra high (MS)
Trace Analysis
well suited
Trace Analysis
not desirable
11

12. Analysis Time/Sample
LC HPTLC
5-60 min / sample
Very slow
1-5 min/ sample
Very fast
1st result -
After 2-3 hours
1st result (̴20 samples batch)-
in 1 hour
Next batch in another 30mins
Next batch in another 30mins
and so on …for whole day
Set-up+set-down time
2-5 hours wasted
Preplanning essential
No set up + set down required
Not essential
12

13. Post Chromatographic Derivatisation
LC HPTLC
Cumbersome
Additional cost
Very simple.
Built into entry level HPTLC
No extra cost.
Limited possibilities
Rarely done
Unlimited possibilities
Every sample derivatizable
1100 known reagents
After
Derivatisation
Before
13

14. Entry Level System Capabilities
LC HPTLC
Capable of
Quantitative analysis
(UV)
Quantitative
(UV, Vis., Fluor.)
+ Spectra, bioactivity, post
chromatographic
derivatisation,
micro preparative
Essential Extras
Degasser
Column oven
water purifier
No
essential
accessories
14

15. Micro-prep Isolation
LC HPTLC
Special
instrumental requirements
Not possible with entry level
configuration
Built in the
entry level HPTLC
2.0mm thick layer only
100 mg sample loaded
2mm thick layer
15

16. Fluorescence
HPTLC
LC HPTLC
Additional detector
required
Additional cost
Built-in the
entry level HPTLC
Every sample can be checked
for fluorescence
Chromatograms with fluorescent fractions Fluorescence scan at 366nm
16

17. UV-Vis Absorbance Spectra
LC HPTLC
PDA detector must
Chromatogram
flowing to waste
Additional cost
Spectrum using
monochromator rotation
Chromatogram
stationary on plate
Built in to
Entry level HPTLC
17

18. Unique Detectors
LC HPTLC
Electrochem Eye
Light scatter In-situ bio-autography
Maximum 2 detectors in tandem 4-6 detectors in tandem
18

19. Chromatography Fingerprint
LC HPTLC
Very Limited
Possibility
Best application
Unique visible info.
Easy to understand
Rarely possible Any complex sample can be
fingerprinted
254nm Visible366nm After Derivatization
19

20. CHROMATOGRAM
Visual Comparison/ Evaluation
LC HPTLC Fingerprint
Not possible Regulatory technique for identification of
botanicals & foods
20
Comparisionof 43 tracks from different
plates/days/labs

21. Lambert’s & Beer’s Law
LC HPTLC
Followed Not followed
Transmission Diffuse reflectance
Dilute solutions Dry layer
21
Large
Linearity range
Short
Linearity range

22. Sensitivity, Precision,
Accuracy, Reproducibility
LC HPTLC
Usually better
but
sample dependent
Can be better
but
sample dependent
Well established methodologyNewly announced methodology
Sample clean up
Very critical
Problematic adds to error
Not critical
Layer disposable
Helps accuracy
MS Detector MS Detector
22

23. Fractions per sample separable
HPTLCLC
~ 10-12~ 15 –30
ISOCRATIC
23
Enough for many samples

24. Separable Fractions per sample
HPTLCLC
Upto50 Upto30
24
GRADIENT

25. Mobile Phase
LC HPTLC
MobilePhase present
during detection
Mobile phase absent
Upto190nm always usable
Limited solvents
as mobile phase
Unlimited choice
Plate disposable
Column life
concerns
Layer disposable
No reuse concern
High volume of expensive
solvents consumed
Small volume of expensive
solvents consumed
100-300 ml/sample 2-4 ml /sample
25

26. Complimentary Aspects
LC HPTLC
RP (C-18) NP (Silica gel)
Partition Adsorption
More retention,
broader peak
More retention,
sharper peak
Rt Rf
26

27. Complimentary Aspects
LC HPTLC
2nd opinion
Final result
1st opinion
Screening
Sample background info.
must before analysis
Not essential.
Helps LC method development
High cost, low speed High speed, low cost
Sample goes to waste Sample recoverable
Can take load off HPLC
where possible
27

28. Limitations
LC HPTLC
Very High pressure Separation bed –6cm
Limited resolution
Sample clean –up must Narrow linearity range
High analysis cost Higher initial investment
High maintenance cost Confused with
Instrumental TLC
Low productivity
Prior info. about
sample is must
Complex, Rigid set-up
Cannot share 6 analysts can use at a time
28

29. HPLC and HPTLC
PART II
Comparison
of
Practical
Considerations
29

30. Most
practical
considerations
favour HPTLC!
30

31. HPLC
Total time -2-4 hrs
Pre-preparation (1-3 hours)
Degas solvents
Set-up parameters
Stabilize baseline
Inject standards
Post analysis (1-3 hours)
Wash column
Set down instruments (1-2 hours)
31
HPTLC
Total time -nil
No pre-preparation
No analysis post preparation
Analysis on demand
Urgent samples welcome
Use W/O planning ahead
Pre & Post Chromatography

32. Maintenance
LC HPTLC
Very high & costly Negligible
Cost x 5 –10 Cost x 1
CMC desirableor AMC +
stock of spares is must
Periodic service
enough
High wear & tear No wear & tear
Problem frequent Problem rare
Downtime
Noticeable
Downtime
Negligible
32

33. Analyst Skills Required
LC HPTLC
Very High
Very veryhigh (LC-MS)
Medium
High (HPTLC-MS)
Very Complex
procedure
Procedure simple,
discreet , stepwise
33
Prior experience essential Not essential
Thorough in
Analytical Chemistry
Familiar

34. Teaching & learning
chromatography
LC HPTLC
Very difficult
Complex,opaque
technique
Accidental damage
possible to LC
Very suitable
Simple
stepwise procedure
Samples visible on plate
Accidental damage
impossible
34

35. Urgent -Important -Unstable samples analysis
LC HPTLC
Own numerous LCs
Or
wait in Q
Analyse on demand
w/o affecting ongoing analysis
1 sample of 1 type
per LC
at a time
Many samples of many types
per HPTLC
at a time
HPTLC -ANALYSIS ON DEMAND.
35

36. Sharing of Chromatograph
LC HPTLC
Impossible Possible
1 channel
chromatograph
6 channels
chromatograph
Machine dedicated
to one sample of one type
Instruments always free.
Samples on plates
Integrated procedure
from start to finish
Time Independent
stepwise procedure
Chromatograph
in a box
Series of instruments
makes a chromatograph
1 or 2 type of
samples analysis/day
10-12 types of samples
analysis/day
36

37. Can “anyone” use hands-on ?
LC HPTLC
No Yes
Can safely use
Accidental damage possible
High pressure
contamination
Analyst uses only key board
Only expert can use Little experience required
Be very careful Quite care-free
37

38. New Method Development & Validation
LC HPTLC
Difficult Simple
Time consuming
30 -45 days
Fast
3 -7 days
High skill Average skill
Expensive
Rs. 2-5 L/ method
Incl. validation
Inexpensive
Rs. 10-50 k/ method
Incl. validation
Engages 1 machine Engages 1/6 capacity
38

39. Possible Output/ Year
New Validated Methods
LC HPTLC
4 -6 40 -50
39
LC HPTLC
3000 -5000 20000 -25000
No. of samples analyzable per chromatograph
Analysis time for one lot of 20 samples
LC HPTLC
Variable
2-10 hrs
Fixed
60-90 min

40. Number of Chromatographs Required
LC HPTLC
Each Dept.
Each Research Group
Each Professor/ Leader
Large number of LCs required
for productivity
One
HPTLC serves
6 analysts / projects
1 –2 enough for productivity
One HPTLC per 10 –15 LCs
is ideal in a lab
40

41. Chromatograph Investment
LC HPTLC
Rs. 25 -40L
(limited capabilities)
Rs. 40 -50L
(extensive capabilities)
World’s best system
essential accessories +5 Lakhs
+ Heavy analysis &
maintenance cost throughout
lifetime.
No essential accessories
Nominal analysis &
maintenance cost in life time
Multiple LC’s
Must keep repeat buying
1 or 2 are enough
Phase wise investment possible
Entry Level System
41

42. Direct cost per sample
LC HPTLC
Very high
X 5-10 (Rs. 250-1000)
Low
X 1 (Rs. 20-100)
Low productivity
5-25 samples/day
High productivity
100-125 samples/day
42

43. Cost of Configurations
LC HPTLC
Entry Level
Lower investment because
of few capabilities
Rs. 35-45L/ world’s best
Entry Level
Higher investment because of
many capabilities
Rs. 40-50L/ world’s best
If LC & HPTLC
have similar capabilities like
Fluorescence, Spectra, Fraction collection, Derivatization
THEN HPLC COSTS MUCH !!
43

44. Revenue Generation Potential
LC HPTLC
No Yes
Commercial analysis for others
is difficult
Commercial analysis for others is
easy
Low throughput High throughout
New methods development &
validation
not so easy
One/ month/ LC
New methods
easy to develop
& validate
5 –10/ month/ HPTLC
Risky to LC No risk ever to HPTLC
No other work possible during
method development
5 channels free for other work
44

45. Best use
LC HPTLC
Batch of
similar samples for final result
analysis
(Pharma QC)
Equally suited for
small or large number of
samples
Machine dedicated to one
analysis
QC and R&D can use
simultaneously
Highest quantification Reliable quantification
Trace analysis Fingerprint of botanicals
High resolution Good Selectivity
45

46. Chromatograph contamination
by sample
LC HPTLC
Possible.
Samples
enters chromatograph
Impossible.
Samples
remain on plate
Sample clean-up
Thorough, expensive,
time consuming.
recovery?
Nominal clean-up
Layer disposable.
Layer can be used for clean-up
46

47. Funds Required
LC HPTLC
High initially Higher initially
Heavy funds required every year
to use and maintain
Rs. 3-5 lakh/year
Little funds required
in life time
Rs. 20-50 k/year
Serves one analysis
at a time
Serves six analysis
at a time
Purchase budget cannot be
shared
Purchase budget shared
between depts/users
Commercial analysis only if
method is known
Commercial analysis of any
unknown sample can be done
Usable life span 7-10 years Usable life 12 –15 years
47

48. Summary-Practical Aspects
LC HPTLC
Very Expensive Moderate Expensive
Lower investment to begin
Heavy during lifetime
Higher initial investment
Nominal later
Low productivity x1 High productivity x 5-10
Cost of analysis x 5 -10 Cost of analysis x1
Maintenance cost x 5 -10 Maintenance cost x 1
Short life 7-10 yrs Long life 10-15 yrs
High skills essential Medium skills
Expert user needed Anyone can use
48

49. Summary-Technical Aspects
LC HPTLC
Highest quantification (±2 %)
For most samples
Adequate quantification(±2 %)
For many samples
Well established
Since 1990’s
Newly recognised
2018
Very high pressure Visual Fingerprint
Very complex & inflexible Negligible expenses
after purchase
Costly to analyse & maintain Very simple to use
Suitable for synthetic
chemistry samples
Synthetic + Botanical samples
Expertise required Post Chrom.Derivatisation
Slowest in lab Fastest in lab 49

50. Best use-LC & HPTLC
LC HPTLC
Quantification of most
synthetic chem. samples
Quantification of
routine samples
Very high resolution Urgent samples
unscheduled samples
High investment
High running cost
Higher investment
Low running cost
One LC dedicated
to one analysis
Share the
chromatograph
High level resource &
expertise needed
Attempt analysis of any
sample
Trace analysis Fingerprint of botanicals
Same sample type,
respective analysis
Different samples
Ideal for Most QC ..Some
R&D
Ideal for Some QC ..Most
R&D samples
50

51. Be Scientific
Think Indian
Be Frugal
Be Practical
USE CAMAG HPTLC FIRST
51

52. THANK YOU!!!
AnchromEnterprises (I) Pvt.Ltd.
52
HPTLC Specialist since 1978!!