Good Chromatographic Practices for Pharmaceutical Industries.

1. Dr. A. Amsavel, M.Sc., B.Ed., Ph.D

2. Content
 Introduction
 Solvents & Reagent Quality
 Mobile phase preparation & usage
 Buffer, Filtration, Degassing
 Storage & usage
 Preparation for analysis
 Sample preparation & System suitability
 Adjustment allowed as per USP
 Column management
 Washing and change of mobile phase
 Analysis and integration
 Documentation
 Problems and reporting
 Qualification and Calibration

3. Introduction
 Chromatography analysis (HPLC, GC & TLC etc ) of material in
pharmaceuticals QC Lab is inevitable
 Chromatographic technique is used for Identification, determination of
assay, purity, related substances and residual solvents etc.
 Purpose of Chromatography analysis is for Batch release, intermediate,
reaction monitoring, process validation, cleaning validation, cleaning
verification, stability testing etc
 Chromatographic analysis has to be performed and documented as per
GMP and to meet the regulatory requirements
 There are challenges, while performing analysis and meeting compliance

4. Qualification And Validation
 HPLC & GC instruments shall be qualified (URS, DQ, IQ, OQ and PQ)
 Software used shall meet the 21CFR Part-11 compliance
 Hardware & Software shall be qualified as per Computer system
validation (CSV)requirement ( GAMP-5)
 Instruments shall be calibrated appropriately at defined interval
 Test method shall be validated, compendia method shall be
verified, and or method transfer as applicable
 Analyst shall be qualified and trained
 Ref :Qualification Of Equipment Annex 1: Qualification Of Liquid
Chromatography Equipment

5. What are the challenges faced while
Chromatographic analysis?
What are the possible errors
occur during analysis?

7. Problems or challenges !!!
How can you prevent the problems?
How can it be controlled?
How errors can be eliminated or minimized?

8.  Follow “Good Chromatographic practices”
 SOP for Good Chromatographic practices
 Qualified Instruments
 Train and Qualify the analyst
 Validated / verified and appropriate test method
 Right Quality solvents, proper sample preparation, system
suitability
 Perform periodic Calibration & Preventive maintenance
Training is focused on HPLC analysis

10. HPLC Analysis Flow

11. Water for HPLC analysis
 Always use only Ultra pure / Milli-Q water for HPLC analysis
 Ultra pure HPLC water of 18MΩ resistivity
 Do not use RO water/de-ionised water for HPLC analysis. It
will have organic and in-organics impurities
 If water contains impurities, it will have higher absorption and
lead to poor baseline , drift , ghost peak and less accurate
Note
 Use fresh water if possible or store properly
 Store HPLC grade water in glass containers.
 Plastic containers may leach the additives in the plastic into the water

12. Solvents & Reagents
 All reagents and solvents should be highest quality.
 HPLC grade reagents & solvents are high purity will have low
UV absorbance
 Low grade solvent contain impurities to produce spurious
peaks, poor peak , high baseline etc
 Ensure that reagent & water used in buffer preparation is of
the highest purity.
 Where required use specific quality of reagent as stated in
Pharmacopiea / test method
 Eg. Different grade of Ammonium acetate based on the purity
 TEA & TFA are expected to be contaminated from the lab
environment and should be refrigerated during storage.

13. Buffers
 Preferably all buffers should be prepared freshly on the day of use .
 This ensures that the buffer pH is unaffected by prolonged storage and
there is no microbial growth; both will affect badly in chromatography.
 If buffer solutions are used for long or stored ; establish the shelf life
and use
 Refer to pharmacopoeia monographs or similar for further guidance.
 Where possible use reagents that contain no stabilizer. Some of the
buffer reagents may contain a stabilizing agent, eg. Sodium metabisulphite.
 These stabilizing agents often may affect the optical and
chromatographic behavior of buffer solutions.
 Avoid the repeated use of solid reagent since may easily contaminate
 Based on use, purchase in low pack size to reduce the exposure.

14. Use of buffer
 Do not leave the buffer solutions for long in system, to avoid
crystallization. This will affect the system.
 Effect on pump - damage plunger and seal
 Effect on column - creation of column voids
 Effect on flow line - corrosion of stainless steel lines
 Possible bacterial growth especially phosphate buffers – it is
good medium for bacterial and fungus growth. Hence, prepared
freshly and avoid storage.
 All the solutions should be clear, homogenous & free from
particulate matter.
 Buffer solutions must be filtered through 0.45µm filter
 Note: small amount of sodium azide to the aqueous solvent to inhibit microbial
growth.

15. Filtration
 All HPLC solvents or mobile phase used should be filtered through a 0.45
µm filter before use.
 Do not keep mobile phase under the suction for more than 5 minutes.
composition of mobile phase change if use highly volatile solvents
 This removes any particulate matter that may cause blockages of column
and system.
 Filtering of mobile phase / solvents will benefit chromatography and the
wear and tear of the HPLC system.
 Pump plungers, seals and check valves will perform better and lifetimes
will be maximized.
 Also column performance and life will be maximised
 Filtered solvents should be stored in a covered reservoir to prevent
evaporation & dust contamination etc.

16. Changing Over of Mobile Phase.
 Changing Over of Mobile Phase one analysis to another
analysis is important for smooth chromatographic analysis.
 Before changing to totally organic phase in the RP system, the
whole system should be flushed with water/organic (1:1)
mixture to remove the buffers used.
 This is to prevent the crystallisation when the organics come
into contact with leftover buffers in system.
 Eg 100% Acetonitrile use will lead to precipitate the salt in the buffer
solution

17. Changing Over of Mobile Phase.
 To convert a normal phase system/column to a reversed phase, flush
with a solvent that is miscible with both the current normal phase
solvents
 If the final reversed phase solvents include a buffer, then it is
advisable to move from the 100% methanol flush to a 50% aqueous
methanol flush.
For example:
Normal phase : Hexane/Ethyl acetate
Flush: IPA (isopropenyl acetate) then Methanol
Finally (50:50) Methanol/Water
Reversed phase : Buffered aqueous methanol

18. Changing Over of Mobile Phase.
 To convert a reversed phase system/column to a normal phase
follow a similar way but in reverse,
 For example,
Reversed Phase : Buffered Aqueous Methanol
Flush :50:50 Methanol/Water
finally Methanol then IPA
Normal Phase :Hexane/Ethyl Acetate

19. Mobile phase preparation- Isocratic
 Different solvents has to be premixed for isocratic systems
 Preparation tips: Measure the solvents separately for accuracy and
mix.
E.g. To prepare 1000ml of 50:50 mixture of water/methanol,
Measure 500ml of milliQ water, transfer into a bottle & transfer 500ml of
Methanol and mix & sonicate for about 10-15 minutes.
 Protection of mobile phase & storage
 Use glass bottle
 Use Plastic bottle for high sensitivity inorganic analysis.
 Always cover the container to prevent evaporation of solvents & dust free
Note: Do not cover the mobile phase bottle with parafilm. The
leachable from the parafilm may contaminate the analysis.

20. Mobile phase preservation & usage
 Do not mix old mobile phase with new mobile phase
 Do not expose the bottle to direct sunlight or wind.
 Use always suction filter
 Keep reservoir above solvent delivery system
 Keep suction filter 2 cm above the bottom to prevent
particulates from reaching pumps
 Periodically clean the suction filter by sonicating in
isopropanol or 1N nitric acid and water
 Filter and sonicate the mobile phase.
 Use mobile phase after reach the room temperature

21. Degassing
 Mobile phase should be thoroughly degassed to remove
all dissolved gasses.
 Dissolved gas can be removed from solution by:
• Bubbling with helium
• Sonication / Vacuum filtration
If the mobile phase is not degassed, air bubbles can form
in the high-pressure system resulting in problems with
system instability, spurious baseline peaks etc.
 Do not use Online degasser while THF as mobile phase
due to degradation of Teflon in vacuum chamber.

22. Advantage of Degassing

23. Suction filter
 All solvent line shall be fitted with an inlet filter to prevent the particles
contamination
 The filters should be kept clean to prevent cross contamination.
 When system is not being used, stored the filters in a solution of 50%
Acetonitrile /50% water and in closed condition.
 This is to prevent the microbial growth, dust and dirt to clog the filter
pores.
 The solvent lines should be clean, clog free, no air bubble and should not
have sharp bends or creases in them.
 The solvent lines and filters should be of sufficient length to reach the
bottom of the solvent reservoir
 Solvent reservoirs should be placed higher than the pump inlet manifold.

24. Equilibration of Column
Column volume:
 This column volume is more correctly called as void volume (Vm).
 The amount of mobile phase required to fill the column.
 Otherway the volume of mobile phase which should be flushed
through a column before it is ready to use
 When a column has just installed on a reverse phase HPLC system
then it will typically require 10 to 20 column volumes.
(exceptions – use of pairing agents & chiral methods)
 This is typically approximately 70%of the total column tube volume.

25. Equilibration of Column

26. HPLC Column Management
 Do not store HPLC columns in buffers. A buffer may precipitate inside the column,
resulting in plugged hits and packing material.
 Mobile phases with 100% or close to 100% buffer may lead to bacterial growth,
which can block the column & frit and packing material.
 Bacteria may also affect your analytes, and organic products from the dead bacteria
may cause "ghost peaks" in chromatograms.
 Do not store HPLC columns in solvents that degrade easily tetrahydrofuran (THF),
triethylamine (TEA), trifluoroacetic acid (TFA).
 Unstabilized THF can form peroxides which may degrade the column
 Make sure that all buffers are washed out of the column before flushing with
Acetonitrile.
 Gradually start the column washing : Eg Starts the flow with 0.2 ml/min and
increases gradually to 2.0 ml/min and continues for 20min to 30 min or as per
procedure
 Have Dedicated Columns for each Method / each product

27. Washing of HPLC System & Columns
 Flush the system thoroughly with strong solvent;
Because switching to pure organic solvent, especially acetonitrile, can
cause buffers or salts to precipitate.
 Recommend switching to non-buffered mobile phase first. Just replace the
buffer bottle with water and run the gradient again.
 Run the gradient to 100% strong solvent (usually methanol or acetonitrile)
and hold it at 100% for 10–15 min to thoroughlyflush the column and
equipment
 Do not shut off a system that contains buffers or salts

28. Washing & Storage of HPLC Columns
 Contamination may change the chromatography or change the column
performance.
 Short term storage, i.e. over night, columns can be stored in the eluent
used in last analysis.
 Middle term storage, i.e. 2 days or over the weekend, columns should be
flushed with pure water to prevent microbial growth.
 Long term storage, silica based columns should be stored in an aprotic
solvent.
 The best storing solvent is Acetonitrile; water content should not be higher
than 50%.
 Buffer salts will precipitate in Acetonitrile and can block the capillaries and
the column.

29. Column cleaning & Regeneration
 Column cleaning has to be used 40 – 60 column volumes appropriate
solvent or as per procedure .
 Check the column efficiency, capacity factor etc. Before and after clean-
up procedure to confirm the improvement of column performance
 Solvent can be used as follows for regenration;
 Normal phase media
1. flush with tetrahydrofuran
2. flush with methanol
3. flush with tetrahydrofuran
4. flush with methylene chloride
5. flush with benzene-free n-hexane
 Reversed phase media
1. flush with HPLC water; inject 4 aliquots of 200 µL DMSO during this flush
2. flush with methanol
3. flush with chloroform
4. flush with methanol

30. System preparedness
 Needle and plunger seals shall be washed before starting the
analysis.
 Use wash solutions, which are miscible with the mobile phase
to wash and plunger seal
 Use separate solutions and containers for plunger seal wash
and needle wash.
 Use of one solution for both functions may compromise the
effectiveness because the functions of these solutions differ.

31. Seal wash - Composition
 Over a period of time small amounts of mobile phase solvents seep through the
seal to the back of the pump head.
 If these solvents contain buffers then the salts may precipitate out forming deposits
which can shorten the life of the seal.
 Seal wash is used when the mobile phase contains buffers.
 It follows that the composition of the seal wash should beaqueous to dissolve
buffers.
 A small amount of organic solvent is added to prevent bacteria growth and also to
reduce the surface tension of the water (this helps the wash solvent cling to
surfaces).
 Typical seal wash composition is 80% water and 20% organic solvent.
 The organic solvent may be methanol, acetonitrile or isopropyl alcohol (IPA)

32. Needle wash
 The needle-wash pump flushes the needle in the sample management
system, preventing carryover of sample between injections.
 The needle-wash also extends the life of the injector seals by removing
buffered mobile phase and sample from the needle.
 The needle in the HPLC system is used to introduce the sample into the
mobile phase so that it can be separated on the HPLC column.
 The needle wash is used to clean the needle after an injection.
 Use “Extended needle wash” from the instrument method if the carry over
is expected, especially during the analysis of Related substances.
 The composition of the needle wash needs to be matched to the sample.
Proportions of aqueous and organic solvents in the mobile phase will be
appropriate.

33. Injector Maintenance
 Purge flow line at least once a day.
 Purge all the flow lines with mobile phase to ensure the solvent tubing is
commpletely filled with mobile phase
 Change purge liquid frequently.
 Never use salt solutions as purge liquid.
 Use septums for sample vial recommended by manufacturer and do not
reuse .
 Do not fill vials to the brim.
 Filter sample with 0.2μm or 0.45μm filters before injection.
 If the sample contains a lot of solid, centrifuge it, and the filter the
solution.

34. Fitting & plumbing
•Estimate the length required.
•Allow extra length if the tubing
is to go around corners as sharp
bends in the tubing will
distort the inside bore
Note: Care should s be taken
when tightening fittings.
Over-tightening can cause
damage to the fitting threads,
ferrules etc, causing the unit to
leak, or can break off in the
housing

35. Guard Columns and or cartridges
 Protect the analytical column from sample and system debris and
contaminants for better column performance and efficiency.
 Guard columns or cartridges are one of the most cost effective and
efficient ways of trapping these unwanted system components.
Note: Guards are designed to be
disposable. Should be replaced once
contaminated
Recommend that the guard is
replaced every 50 – 100 injections or
based on user

36. Precaution
 Avoid pressure shocks on the column.
 Pressure shocks lead to channeling in the column, which
results in peak splitting in corresponding chromatogram.
 Always keep both ends of the column closed, after usage.
 Keep the columns in the designated column cabinets after
use.
 Use the pH range of 2 to 8 or follow the manufacturers
instructions

37. Sample Preparation
 Sample preparation is not only dissolution of a solid in a liquid.
 If Samples require other techniques such as filtration, extraction or derivitization ,
perform properly
 Accurate weighing and/or dissolution or make up volme for assay.
 Filtrate the sample if it contain suspended solids. By syringe filter or using on-line
pre-column filter
 Sample solvents should match the mobile phase to avoid baseline errors and
spurious peaks
 The sample is clean the longer the life of column and reliable
 If sample contaminates the column, we may spend spend more time & money
 Important: If using a membrane filter make sure that it is compatible with all the solvent used

38. Definition
 Dwell volume (D): Also known as “gradient delay volume”, is the volume
between the point at which the eluents meet
and the top of the column.
 Hold-up volume (VM): The volume of mobile phase required for elution of
an unretained component.
 Resolution (RS): The resolution is the separation of two components in a
mixture,
 Retention volume (VR): The volume of mobile phase required for elution
of a component (Vm= retention time (TR) X flow rate (F)
 Separation factor (α): The relative retention calculated for two adjacent
peaks (k2/k1)
 Theoretical plates (N): A measure of column efficiency based on RT & PW

39. System Suitability
 System suitability test shall be performed to verify that the
chromatographic system is adequate for the intended analysis.
 SS performance is based on the equipment, electronics, analytical
operations, and samples analyzed etc .
 The following factors may affect chromatographic behavior;
 Composition, ionic strength, temperature, and pH of the mobile phase
 Flow rate, column dimensions, column temperature, and pressure
 Stationary phase characteristics- type of column (particle-based or
monolithic), particle or pore size, porosity, and specific surface area
 Reverse-phase and other surface modification of the stationary phases, the
extent of chemical modification (as expressed by end-capping, carbon
loading, and others)

40. System Suitability Parameters
 Resolution between specified peaks ( >2)
 Theoretical plate (N>2000)
 Precision / injection repeatability : % RSD of replicate injection unless
otherwise specified
 % RSD for five replicate injections of the analyte- ≤ 2.0%
 % RSD for five replicate injections of the analyte- > 2.0%
(Can vary based on the purpose assay , RC, etc from 0.7% to 10%)
 Tailing factor (T <2)
 Capacity factor ( k' > 2 )

41. Resolution
 Resolution is defined as the distance between two adjacent peak
apexes, divided by the average base width of both peaks.
 Resolution is dependant on three other variables, the column
efficiency (N), capacity factor (k’) & selectivity (α).
 Increasing N increases resolution because peak width decreases.
Decreasing N decreases the resolution due to broad peak width
 Decreasing k’ sharpens the peaks but decreases resolution.
Increasing k’ broadens the peaks but improves resolution.
 Increasing α increases resolution. One peak moves relative to the
other. Likewise, decreasing α decreases resolution.

42. Capacity factor
 Capacity factor (N) is affected by changes in mobile phase, operating
temperature, analyte retention characteristics and changes to the surface
chemistry of the column.
 Changes in N that occur both with standard and sample mixes are likely to
be due to changes in the column, temperature or mobile phase
composition.
 Changes in N that occur only in the sample mix and not the standard mix
are most likely to be due to the composition of the sample.
 Note: Capacity factor will change by up to 10% for a 5°C rise in column
temperature.
 Selectivity is a measure of the relative retention of two adjacent peaks
in a chromatogram

43. Band spreading
 Broad peaks with change in retention time, indicate band spreading.
 It can occur within the HPLC column or due to incorrect system
plumbing.
 Follow the below to measure the band spreading due to the HPLC
system.
Remove the HPLC column from the system and replace with a zero
dead volume union. Inject column efficiency solution and calculate
 Band Spread (µL) = Peak Width x (1/20) x 1 x 1000
Confirm column effects or plumbing .

44. Solvent property
 Understand and cerify the solvent property which may affect the
HPLC analysis
 physical properties, like viscosity and miscibility number. Are
significant property
 The miscibility numbers can be used to predict the miscibility of
solvents.
 If the smaller miscibility number is subtracted from the larger and
the difference is 15 units or less, then the two liquids are soluble in
all proportions at 15°C.
 Difference in Smaller miscibility number is < 15 mixer of solvents
lead to poor chromatographic analysis and less accurate.

45. Solvent properties

46. Impact of PH
 In reversed phase HPLC, the retention of analytes is related to their
hydrophobicity. The more hydrophobic the analyte, the longer it is
retained. When an analyte is ionized, it becomes less hydrophobic
therefore its decreases.
 When separating mixtures containing acids and/or bases by
reversed phase HPLC, it is necessary to control the pH of the mobile
phase of buffer in order to achieve reproducible results.
 Eg Resolution changes to 1.4 from 3.0 due to less of 0.1pH

47. Adjustment allowed for HPLC condition
Property USP General Chapter 621 Ph.Eur. Gen. Chapter 2.2.46
Column length ±70% ±70%
Particle size Reduction by 50% Reduction by 50%
No increase No increase
Internal diameter Can be adapted as long as the linear flow
velocity remains the same
±25%
Flow rate ±50% or more, provided the linear flow
velocity remains the same
±50%
Column temp. ±10 °C ±10 °C, maximum 60 °C
Injection volume Reduction allowed as far as precision and
detection limit acceptable. No increase.
Reduction allowed as far as precision and
detection limit acceptable. No increase.
pH -mobile phase ±0.2 units ±0.2 units (±1% for neutral subs)
Salt concentration of
the buffer
±10%, as far as the allowed change in pH value ±10%
Composition of
mobile phase
Minor components ±30%, if not more than
±10% absolute
Minor components ±30%,if not more than
±2% absolute (greater value accepted)
Wavelength Not permitted , can be Max ±3nm based on
the validation
*For gradient separation, a change of the mobile phase is not recommended

48. Starting HPLC analysis
 Test method shall be created and verified
 Sequence can be as below or as per test method
 Blank, system suitability, reference solution , test
bracketing standard
 Data file shall be continuous (do not repeat)
 Load the sample and run the system suitabilty
 After SS passes run the test
 After completion process the chromatograms
 Print the method, sequence and data
 Perform audit trail store and keep data back up

49. Integration
 Do not integrate any peak manually.
 Integrate all the sample sets batch wise.
 Always use same processing method for processing of
blank, standard & sample chromatograms in case of Assay
& related substances, etc.
 Verify the processing parameters like
 Threshold,
 Width,
 System suitability,
 Peak names etc.
 Save the processing method

50. Integration
 Integrate all the injections including entire set of failures.
 For System suitability injection for which Tailing factor,
Resolution or Theoretical plates are to be checked,
consider only the first injection of the sequence.
 Note : Do not consider this criteria for average of all the standard
injections unless otherwise specified.
 Re-integration:
 Do not re-integrate the chromatograms without
documenting.
 Document reason for reintegration.

51. Common problem in HPLC analysis
 How to handle the failure ?
 SOP shall be available and shall address the handling of Lab deviation/
incidents.
 SOP shall define clearly the deviation/ incident , reporting investigation,
CAPA and documentation
 Record all the deviation/ incident happened in chromatographic analysis
 Process all the injections including the invalid injection and report and
store the data along with Raw data.
 Do not omit any injection
 Investigate the deviation/ incident
 Find the root cause for the sample set failure.
 Rectify the problem, take appropriate CAPA and document
 Repeat complete sample set of injections in case of sample injection failure

52. Common problem in HPLC analysis
 System failures may occur during analysis due to
 System over pressure
 Communication error
 Failure of system suitability
 Peak splitting/ negative peak
 Bracketing standard failure

53. How to handle the problem if any
 In case of interruption, due to power failure, computer
interruption, time gap due to sample preparation or due to
injection for ~4 hours and if system is in continuous state of
equilibrium ;
 Inject bracketing standard and proceed otherwise restart
 Deviation in RT for sample or std, is > 15% of specified RT
 Inhibit the peak upto void volume
 Use the same integration parameter for entire set
 Reprocess shall be at the same time for entire sequence

54. Documentation
Ensure are followed contemporaneously
 Instrument Use Log
 Routine Maintenance Log
 Problem Log
 Column History Log

55. PQ of HPLC
Component Parameter Acceptance Criteria
Pump Pump Flow Accuracy 0.5ml (0.475 to 0.525) 5.0ml (4.75 to 5.25)
Pump flow precision RT % RSD:NMT:0.50
Gradient composition in % 20, 40, 60, 80 + 2.0
Column oven Column Temperature Accuracy Column Oven: < 2.0
Column Temperature Stability Column Oven: < 1.0
Sample oven Sample temperature Accuracy Set temperature 4°C: > -2.00 and < 5.00
UV Detector Wavelength Accuracy (201nm to 209nm) 205 nm < 2
(241nm to 249nm) 245 nm < 2
(269nm to 277nm) 273 nm < 2
Noise and Drift Noise: < 0.040 mAU Drift: < 0.500 mAU/Hr
Signal to Noise > 3000
Response Linearity (Resp.Factors) Correlation coefficient: > 0.99900
Lamp Intensity 1000
Sampler /
Injectors
Injector Precision
Volume Delivery - Linearity
% RSD for Area: < 1.0 % RSD for Height: < 2.00
Correlation coefficient: NLT:0.99
Injection Carryover Carryover for Area: < 0.20
Carryover for Area: < 0.40
Ensure the below as parameter during PQ as min, but not limited to;

56. Calibration of HPLC
Component Calibration test Acceptance Criteria
Pump Leak Test No leak
Flow rate Accuracy 0.5ml (0.49 to 0.51)
1.0ml (0.98 to 1.02)
2.0ml (1.96 to 2.04)
Flow stability RT % RSD:NMT:1.0
Gradient Delivery Accuracy in % 20, 40, 60, 80 ±2.0%
Column oven
&Sampler
Temperature Accuracy by Calibration of
Thermocouple and Air temperature
Column Oven:
25°C/40°C/60°C + 2.0
UV Detector Wavelength Accuracy(266nm to 276nm) 271 to 273nm
Dynamic Short-term Noise (Single to Noise
Ratio)
Noise:0.04 mAU or less
Drift:5.0mAU/hr or less
Response Linearity Correlation coefficient: NLT:0.99
Lamp energy Low intensity (> 200)
Average intensity (> 5000)
Highest intensity (> 10000)
Sampler /
Injectors
Volume Precision % RSD :NMT 1.0
Volume Delivery - Linearity Correlation coefficient:NLT:0.99
Injector Carryover NMT 0.1

57. Reference
 WHO 791 Guideline for GOOD CHROMOTOGRAPHY
PRACTICES (February 2019)
 Troubleshooting LC Systems, John W Dolan & Lloyd R
Snyder, Humana Press
 Qualification Of Equipment Annex 1: Qualification Of
Liquid Chromatography Equipment

58. Thank you