The Determination of 6 Gingerol In

Local and Imported Ginger Rhizomes

By HPLC

By
K. Thomas
T. Green
R. Wiggan
History and Uses of Ginger Rhizome

Ginger has been used in China for the past 2500 years

Greeks and Romans used ginger f...
Structure of Ginger Rhizome

/  Outer layer of Cork cells

‘. -
_'_-

   
   
  

 

Inner layer of radially arranged cork...
Chemical Constituents of Ginger
Rhizome

gingerols

shogaols

paradols
5% zingiberene
10% AR-curcumene
10% beta-sesquiphel...
Main Pungent Principles Of Ginger
Rhizome

Bitters (bitter principles) are a heterogeneous group of naturally occurring
co...
Properties of [6]-gingerol

~ Appearance :  Yellow oily liquid
- Molecular Structure: 

OH O

OH
5-li_vdmxy- I -(4-hydrnx_...
Laboratory Synthesis of [6]-Gingerol

OMe
[6]-Gingerol on
Bond to be formed
Aldehyde must by aldol macho" Ketone must not
...
Laboratory Synthesis of [6]-Gingerol

9
cont d
Step 1: Protect Hydroxy Group

0 0
We one
. lc~, Si(‘l
 »
OH Et-, N
OSIVE3
...
Laboratory Synthesis of [6]-Gingerol

9
cont d
Step 3:Protect Carbonyl by substituting
SiMe,  for Li
OLI
OS‘-. ’>3;, 
Ol-l...
Properties of [6]-Shogaol

‘ Molecular Structure: 

1-(4-hydroxy-3-methoxyphenyl)decan-4-ene-3-one

* Soluble in methanol
...
6-Shogaol

1 "
I l
__
, —- -"
. ..p
l air
. .."‘,  ‘
‘/  n‘. 
«V
t. 
.a
,  .. rq, . . v«
Nonanoic Acid Vanillylamide
(External Standard)

° White powder

_J’

wt

,  gs. )

° Soluble in methanol ‘ re  -. _f*.  «...
Validated Standard Method(s)

‘ 1.Ginger and its Oleoresins-Determination of the
Main Pungent Components-Method Using High...
Apparatus ( extraction)

1. Volumetric Flasks (10 ml,  25 ml ,50 ml,  100ml)
2. Pipettes (5 ml,10ml )

3. Filter funnel

4...
EXTRACTION OF GINGER
OLEORESIN FROM GINGER
RHIZOME
Step 1:S1ice ginger rhizome into
Slices approximately 1cm in diameter
Step 2:Weigh sliced ginger rhizome

* nsi’;  O‘? 
‘jg 2 “ H . .
. M _

7.1 "V_ .  ”~ ii";  .
.  - "5 3::  ,2 ~ ’ gar -"tits
.  - ' l , . I.   ‘ I ,  ‘M
.  ‘ -1-.  - V  ,  - " ' .  I , .«...
Step 4: Stir methanol/  ginger mixture
for 1 hour
Step 5:Filter Mixture

;  '“1;—" I  y~; ;.v '<r. v.. ... ,-“,2 . 
1:5 um:  I , 
 ,  _ . . _ -3'-'12:» -
1 _ ‘ I - l  ‘:26;...
Step 6: Weigh empty round
bottom flask
4" . ... .». :@.     r ‘J -, 'v .  ' xx
 I,   ‘yYI“ w“ _ I g I‘ ,  . . /~, v &_ I .  1_“‘
.  ii‘;  : - .  _‘ Iv. » .  .b _...
Step 7: Evaporate methanol from
the methanol oleoresin mixture using
the rotary evaporator.
Rotary 

1
StCp 83 Dissolve oleoresin in flask in methanol,  filter into
a 100ml volumetric flask and reweigh R. B flask after transfer

'
Step 9: Prepare External Standard
Nonanoic Acid Vanillylamide

Weigh 0.85g of NVA

Dissolve sample in 100ml methanol in 10...
Preparation of Sample for HPLC
Analysis
1.Filter All Solutions
To Prevent Blockage of Column

(Sample Solutions,  Standards and Solvents)
ii 3Filtration Apparatus
Clamp 5 l

I
I

~,  Glacial accti, ‘  i‘

 

E
I E-J. ,t"If| lInI

“ . _‘ — ‘<3 ‘I

-3; . ‘ . _ 
...
9}.  ‘llil'21ll(_)ll. «‘. '1’? :‘i'1lillS
l
l

l

I
I

Mobile Phaseil at “er.  .
Aceton vi . -
2.Degas All Solutions
(Sample Solutions,  Standards and Solvents)
on
". 

H‘,  _

. _.4-. ._

    

Degassing
 Apparatus
 "xv l

“f i 11!‘; 

—. ''-H, 
l‘  . ,

_ V. ‘

li. ‘ ll . ,

(JKE¢...
. . .m .  .5. . 
, l
u ' n lllllll l l . 'l.  M

.  M.  u

9.. 
ll . 
_ E
. 
A _
.  .1
I(| .l ll
‘ .  I ll

nflxxja
Instrument Parameters

Instrument Instrument Settings
Pararneterslconditions

woorozzom
Mobile Phase:  Isocratic Acetonitr...
:1

: )1

ill‘

ill‘
Specifications

Detector

Stationary Phase:  Guard
Column

Stationary Phase:  at analytical
column

Specifications

Waters H...
Calculations

l. Mass of ginger rhizome used

2.Mass of ginger oleoresin extracted

3.Mass of ginger oleoresin dissolved

...
Determination of Mass of Ginger
Oleoresin Extracted From Fresh
Ginger Rhizome
The Extraction of Ginger Oleoresin from Fresh Ginger

Rhizome

Mass of R. B flask +residue before transfer — l64.27g

Mass ...
External Standard Nonanoic Acid V anillylamide

 lfl9L

Muss of NVA :  0.0ls‘5g (85.()mg)

Volume of methanol used :  I00 m...
Blank Methanol

0.0006
0.0004
0.0002
0.0000 1
-0.0002

-0.0004
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22....
AU

Nonanoic Vanillylamide 0.17 mgml"

0.0015

0 005

0004

0.003

0.002

0.001

0.000

2.00

400

6.00

800

10.00

12 00...
AU

0.0020
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004

I
0.0002

0.0000

Nonanoic Vanillylamide 0.34 mgml"

2...
AU

0.040
0.035
0 030
0.025
0.020
0.015
0.010
0 005
0.000

200

Stock NVA O.85mgm| "

4.00

6.CI)

8.00

, _-/  NVA peak
....
Ginger Oleoresin
Chromato grams
0 0203

0 0302

0 0301

0 -C000

-0 C001

-0 CW2 1

200

Blank Methanol

4.00

6.00

800

10 C0
1.1n. ‘.es

1200

14.00

1...
D
<(

Ginger Oleoresin( 6.4 rngml")

0.00035
0.00030
1 . _—(, :|"‘:  ‘mil 1‘ ‘ ‘
0.00025  K»
; _. g«’”*. ’_'*§fi , .

0.000...
Ginger Oleoresini 12.8 rngml“)

0.0305
x«. ;;:   
0.0304 1 1 ‘ ‘l L
: fl__»§_. “‘: ’~“‘f;1,
0.0303 5 0' :5 
0.0302
0.0301 »...
New Stock Ginger Oleoresin 52.0
mgm| "‘

0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000

2.03 4.00 6.00 8.00 1000 1...
Detection Limit of UV/ V is Detector

Blank (Methanol) NVA St°°k

Lir»: -ar Plat cl [N‘‘ALxi'Ab: c-11:1»:  :2

E‘ JCC -C2
...
Detection Limit of UVNis Detector

Bhnk (Methanol) Stock ginger Oleoresin 12.8 mgml"

    
 

Llnult l‘| at aO| ?lVA[ vu A...
0.85

Table of Data Obtained from HPLC

Analyses

. «‘miwto5 Arc-3

Cmc-antration

.5
*3.

Ahsorba were at maximum peak

-...
Calculations of Response Factor

The response factor for NVA is calculated as follows: 
K“,  = (Cm x 100)I, ,., ,, mgI100m...
Response Factor for 6-Gingerol

K[6]-G =  KNVA x 298,38I293,41
=  KNVA x 1,003 mgl100m| lunit area
=6.00E-02 mgl100m| luni...
Concentration of 6-Gingerol in 52
mg/ m101eoresin Solution
- Calculated using:  (AMG XKMG)/ C

C= concentration of ginger ...
Error Analysis

Equipment Tolerance
Analytical Balance 0.00059

Sml Pipette 0.05mI

10ml pipette 0.04mi

10ml volumetric f...
Error Analysis Continued

Error in mass of ginger oleoresin " (0.0OO53+ 0.0005"
=7.071x10‘5g =  7.071 x1 0"mg

Error in co...
Sample Storage

* Nonanoic acid Vanillylamide is
thermally labile NVA was refrigerated
when not in use. 

-' Ginger oleore...
Requirements

* Spike the sample and perform extraction in
duplicate —Insufficient sample

* Concentration of 6-gingerol

*...
Methods used to Ensure Data Quality

* Blanks
* Used validated standard method
* Extemal standard
#0-

Problems

Technical difficulties associated with
HPLC

Fluctuating pressure, 
solvent, baseline, curcumin, backflow o...
.°": l‘. °°! ’. -‘

Deviations from Validated
Standard Method

Concentration of NVA standard
Mixing time

Concentration of...
Suggestions

* Water trap should be attached to the
degassing apparatus

* ISO 9001 protocols regarding cleanup, 
definiti...
Conclusions

° Of the results obtained,  the values generated
for the percentage mass of ginger oleoresin
were of the high...
Part II:  Organic Components
in Ginger by UVNis
Spectroscopy
Extraction Method

. Due to lack of time and availability of
Samples the previously prepared ginger
oleoresin was used
Trial 1:Vis/ Nir Spectrophotometer

Pu8670 Vis NIR spectrophotometer

 

- , -_,  ., m  L1,-
ru'I" 
-. '': V . ’.‘'. I ’ '...
Instrumental Parameters

Ir Wavelength -325nm

* Response linear with respect to ginger
oleoresin

Ir NVA below the detect...
Trial 2:UV/ Vis Spectrophotometer

Spectronic 601 UVNis Spectrophotometer

,1 _
‘ I
it
t
W’ . _ . 
__-4/0 _i’£0,'_2", 
‘  ...
Instrumental Parameters

* Wavelength -282nm
'* Cuvette- Quartz
* Path length -lcm
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C30J 6 Gingerol

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  • l.o.l Had a problem when I was there where someone reused my stuff word for word,the point of this is just to give an idea,nobody from uwi posts their presentations,which is a pity,because there are some pretty good ones.This has been on here for over 3 years.I was sorta hoping others would catch on and start posting their stuff online,but noone has.
    Ie web 2.0 sort of thing,plus to give others a roadmap,ie that the stuff has been done before etc.I'm honestly surprised gingerol is still relevant,I thought they'd have new stuff by now l.o.l
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C30J 6 Gingerol

  1. 1. The Determination of 6 Gingerol In Local and Imported Ginger Rhizomes By HPLC By K. Thomas T. Green R. Wiggan
  2. 2. History and Uses of Ginger Rhizome Ginger has been used in China for the past 2500 years Greeks and Romans used ginger for its therapeutic value ranging from stomach upsets to nausea and vomiting The use of ginger in the kitchens as a condiment came about in the 13th century. Its gastrointestinal folk uses have been as stomachic, purgative. antispasmodic, appetizer and in diarrhoea. anorexia. colic. dyspepsia. biliousness. vomiting. condiment. sialagogue and in toothaches The toxicity of ginger is generally considered to be negligible hence its use as a common spice and food
  3. 3. Structure of Ginger Rhizome / Outer layer of Cork cells ‘. - _'_- Inner layer of radially arranged cork cells Outer zone of cortex composed of flattened cells Oil cells Endodermis Zone of small vascular bundles Vascular bundle "*-. t Fibrous sheath of vascular bundle
  4. 4. Chemical Constituents of Ginger Rhizome gingerols shogaols paradols 5% zingiberene 10% AR-curcumene 10% beta-sesquiphellandrene 8% bisabolene 6% dextro-camphene 3% beta-phcllandrcnc 2% 1,8-cineole citral-a (geranial) citral-b (neral) alpha-pinene myrcene
  5. 5. Main Pungent Principles Of Ginger Rhizome Bitters (bitter principles) are a heterogeneous group of naturally occurring compounds. marked by their strong bitter taste and therapeutic importance rather than a chemical classification. "Bitterness value" is a rating system based on the amount of water in which a one gram extract of the compound can be diluted into and still retain a bitter taste. [6]-Gingcrol is responsible for the characteristic taste of ginger
  6. 6. Properties of [6]-gingerol ~ Appearance : Yellow oily liquid - Molecular Structure: OH O OH 5-li_vdmxy- I -(4-hydrnx_v—3-niethnxyphenyl ldecan-3—onc ' C17H26O4 - M. P (Crystalline form) 30 C - 32C - Mol. Wt. : 294.3859 ° Soluble in methanol, acetonltrile, ether, chloroform, benzene - UV absorption : ~ 282nm — 284 nm _¥_ 94 , : a’ ‘’'i‘ . e , is-~~‘. ‘¢'f‘ _ , _ L. £:. §u‘ 04" Q "4 Chiral Chiral center at C-O-H R and S enantiomers
  7. 7. Laboratory Synthesis of [6]-Gingerol OMe [6]-Gingerol on Bond to be formed Aldehyde must by aldol macho" Ketone must not act as electrophile hee‘fl :3 act as electrophile here """"" ‘; / / Cl'. tle Aldehyde must Ketone must not - Ketone must not enolize here - enolize here enolize here
  8. 8. Laboratory Synthesis of [6]-Gingerol 9 cont d Step 1: Protect Hydroxy Group 0 0 We one . lc~, Si(‘l » OH Et-, N OSIVE3 -l-{-l-liydro. xy-3~nicthox}q)hcnylibutun-2-one Step 2:Protect Carbonyl Group G [Ll 0lJ Ckle llr: ,Sl—- N C‘-. 'e 5i‘. le3 Tb C-S 'i. le, CS kit";
  9. 9. Laboratory Synthesis of [6]-Gingerol 9 cont d Step 3:Protect Carbonyl by substituting SiMe, for Li OLI OS‘-. ’>3;, Ol-li: iii. -.. slcI 0”“ osime, OS ‘vie; Step 4:A| doI Reaction osiiie, 0 Olllc / /)b on 0 ‘mini' H 0"‘ °5"‘°' Tm‘ [6]-G~‘nut—riil ' on 927-‘; Yield
  10. 10. Properties of [6]-Shogaol ‘ Molecular Structure: 1-(4-hydroxy-3-methoxyphenyl)decan-4-ene-3-one * Soluble in methanol ' Formed by heating 6-gingerol at low pH
  11. 11. 6-Shogaol 1 " I l __ , —- -" . ..p l air . .."‘, ‘ ‘/ n‘. «V t. .a , .. rq, . . v«
  12. 12. Nonanoic Acid Vanillylamide (External Standard) ° White powder _J’ wt , gs. ) ° Soluble in methanol ‘ re -. _f*. «*4, / ,4 4, J tr -2 H ° Irritant to skin and eyes
  13. 13. Validated Standard Method(s) ‘ 1.Ginger and its Oleoresins-Determination of the Main Pungent Components-Method Using High Performance Liquid Chromatography ISO Reference# 13685: 1 997 * 2. Bailey-Shaw , Y. et a1,Supporting the Development of A Nutraceutical Industry in . lamaica- SRC Research Report,2003,pp 20-24
  14. 14. Apparatus ( extraction) 1. Volumetric Flasks (10 ml, 25 ml ,50 ml, 100ml) 2. Pipettes (5 ml,10ml ) 3. Filter funnel 4. Buchncr Funnel 5. Filter Flask 6. Whatman Number 4 grade filter paper 7. Round bottom flask (1001n1) 8. Rotary Evaporator 9. Blender 10. Knife
  15. 15. EXTRACTION OF GINGER OLEORESIN FROM GINGER RHIZOME
  16. 16. Step 1:S1ice ginger rhizome into Slices approximately 1cm in diameter
  17. 17. Step 2:Weigh sliced ginger rhizome * nsi’; O‘? ‘jg 2 “ H . .
  18. 18. . M _ 7.1 "V_ . ”~ ii"; . . - "5 3:: ,2 ~ ’ gar -"tits . - ' l , . I. ‘ I , ‘M . ‘ -1-. - V , - " ' . I , .«'‘. ‘-’. ~'‘’'‘ I. in‘ , K " I . fix’ -‘z~, s—»i~‘, «*-$3 { H U’ : ) €. .. ‘I F - ' com/ — _ ks . = '; ."" J. '1 I '1" I‘ I ‘ r‘'‘'. ..Tfi‘: g)‘; V-: I‘’ 5 — f 1? I — Lian ‘ " __. ,4 {', .. -:»' i‘_' , , . . 1: V U Step 3: Blend ginger rhizome with 250 ml methanol ‘fr "1 ’ '. :;-. : ' * --—. *_~: .. ..-no. >7 ; .l . f’ I t’ .5 l {I '- . ' f" I‘ i F7 ‘V: ' l. — P
  19. 19. Step 4: Stir methanol/ ginger mixture for 1 hour
  20. 20. Step 5:Filter Mixture ; '“1;—" I y~; ;.v '<r. v.. ... ,-“,2 . 1:5 um: I , , _ . . _ -3'-'12:» - 1 _ ‘ I - l ‘:26; . . I I p = ; 1 l§“ p . IQ ' n
  21. 21. Step 6: Weigh empty round bottom flask
  22. 22. 4" . ... .». :@. r ‘J -, 'v . ' xx I, ‘yYI“ w“ _ I g I‘ , . . /~, v &_ I . 1_“‘ . ii‘; : - . _‘ Iv. » . .b _V f I "‘ . A-4‘ . . , .. .4 4~ ~ 1‘ _. _, " $3 . '_. 4-, ‘ » I _ . , . ,t__"' fig M - , r_ I . ‘ ’ _-‘ ‘. , . ,1 , .A, _¢. a-3"‘ ‘ . if F‘ . “' I —. }‘/ FF . . ~ ; W . - " “=2 ‘ J 1 K‘ ‘ - . .~"“ ’"' -mt“-° _ gr». : ‘ ? -‘'§-_. , s; ~.f. ‘ V ' S » E 'Round K I Bottom K—-I Flask(100ml)
  23. 23. Step 7: Evaporate methanol from the methanol oleoresin mixture using the rotary evaporator.
  24. 24. Rotary 1
  25. 25. StCp 83 Dissolve oleoresin in flask in methanol, filter into a 100ml volumetric flask and reweigh R. B flask after transfer '
  26. 26. Step 9: Prepare External Standard Nonanoic Acid Vanillylamide Weigh 0.85g of NVA Dissolve sample in 100ml methanol in 100ml volumetric flask Prcparc secondary standards by pippctting Sml and I0 ml aliquots into separate 25ml volumetric flasks J:
  27. 27. Preparation of Sample for HPLC Analysis
  28. 28. 1.Filter All Solutions To Prevent Blockage of Column (Sample Solutions, Standards and Solvents)
  29. 29. ii 3Filtration Apparatus Clamp 5 l I I ~, Glacial accti, ‘ i‘ E I E-J. ,t"If| lInI “ . _‘ — ‘<3 ‘I -3; . ‘ . _ . g , I k‘ I Mobile Pliasei. :)l “er . Distilled Wat iii
  30. 30. 9}. ‘llil'21ll(_)ll. «‘. '1’? :‘i'1lillS l l l I I Mobile Phaseil at “er. . Aceton vi . -
  31. 31. 2.Degas All Solutions (Sample Solutions, Standards and Solvents)
  32. 32. on ". H‘, _ . _.4-. ._ Degassing Apparatus "xv l “f i 11!‘; —. ''-H, l‘ . , _ V. ‘ li. ‘ ll . , (JKE¢I1]: “‘ If . '1 ~. . » (uL ll. ‘.. tl''3«-. ) 1.’l’l; ‘.‘ 1..
  33. 33. . . .m . .5. . , l u ' n lllllll l l . 'l. M . M. u 9.. ll . _ E . A _ . .1 I(| .l ll ‘ . I ll nflxxja
  34. 34. Instrument Parameters Instrument Instrument Settings Pararneterslconditions woorozzom Mobile Phase: Isocratic Acetonitrilezwater 65:35 Flownate Stationary Phase: Al Guard Column Ultrasphere -ODS Rp-C-8 10.0pm (25mm x 4.6mm 1.D) Stationary Phase: At Analytical Ultrasphere-ODS Column RP-C-8 5.0 pm (15 x4.6 mm 1.D wavelength on UV detector Analysts 1”lme
  35. 35. :1 : )1 ill‘ ill‘
  36. 36. Specifications Detector Stationary Phase: Guard Column Stationary Phase: at analytical column Specifications Waters HPLC 600 Waters prep LC 4000 Waters 486 tuneable absorbance detector Ultrasphere-ODS R. P(guard column) C8 10 micrometer (25 x 4.6 mm | .D) Ultrasphere-ODS R. P(guard column) C8 10 micrometer (15mm x 4.6 | .D)
  37. 37. Calculations l. Mass of ginger rhizome used 2.Mass of ginger oleoresin extracted 3.Mass of ginger oleoresin dissolved 4.Concentration of stock solution of ginger oleoresin : 5.Percentage of ginger oleoresin in sample.
  38. 38. Determination of Mass of Ginger Oleoresin Extracted From Fresh Ginger Rhizome
  39. 39. The Extraction of Ginger Oleoresin from Fresh Ginger Rhizome Mass of R. B flask +residue before transfer — l64.27g Mass of R. B flask +residue after transfer = 163.63g Mass of ginger oleoresin transferred — 164.27g - 163.63g = 0.27g Volume of methanol added — 50 cm’ Mass Concentration of gingerol stock solution — 0.27g . / 50cm’ =0.0l 28 gem‘-‘ Mass ofR. B flask +residue before transfer — l64.27g Mass of empty RB flask — 163.23g Mass of oleoresin extracted from original sample = 164.27g -l63.23g = l.04g Mass of fresh ginger = 55.820g Percentage oleoresin by mass -I .04g/ S5.820g x 100% =1 .863"/ o
  40. 40. External Standard Nonanoic Acid V anillylamide lfl9L Muss of NVA : 0.0ls‘5g (85.()mg) Volume of methanol used : I00 ml Mass concentration of stock solution 85.0mg 100cm‘ : 0.85 mgcm'-‘ Semndarxstandaulfiolilfinnssztlilia Solution Number Stock Required Actual Concentration concentration concentration ""8 91"" l'mg cm ‘ fmg cm ‘
  41. 41. Blank Methanol 0.0006 0.0004 0.0002 0.0000 1 -0.0002 -0.0004 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Mnutes
  42. 42. AU Nonanoic Vanillylamide 0.17 mgml" 0.0015 0 005 0004 0.003 0.002 0.001 0.000 2.00 400 6.00 800 10.00 12 00 M wutes 14.00 16 0'3 1800 2000 22.00 2400
  43. 43. AU 0.0020 0.0018 0.0016 0.0014 0.0012 0.0010 0.0008 0.0006 0.0004 I 0.0002 0.0000 Nonanoic Vanillylamide 0.34 mgml" 2.00 4.00 6.00 8.00 10.00 . )—J' .4” *1.‘ :2‘ 12.00 Mnutes 71 »4 Q .7’ -. <0 1 .1 J. I I J»? 14.00 16.00 18.00 20.00 22.00 24.00
  44. 44. AU 0.040 0.035 0 030 0.025 0.020 0.015 0.010 0 005 0.000 200 Stock NVA O.85mgm| " 4.00 6.CI) 8.00 , _-/ NVA peak . . r. ‘ / ‘— , <‘. j 12.00 14 00 10.00 M wutes 16 CD 18.00 20.00 22.00 24.00
  45. 45. Ginger Oleoresin Chromato grams
  46. 46. 0 0203 0 0302 0 0301 0 -C000 -0 C001 -0 CW2 1 200 Blank Methanol 4.00 6.00 800 10 C0 1.1n. ‘.es 1200 14.00 1600 18 00 20.00
  47. 47. D <( Ginger Oleoresin( 6.4 rngml") 0.00035 0.00030 1 . _—(, :|"‘: ‘mil 1‘ ‘ ‘ 0.00025 K» ; _. g«’”*. ’_'*§fi , . 0.00020 r”—' ‘~"‘: z'I~, 0.00015 0.00010 0.03005 0.03000 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 13.00 Mnutes 20.00
  48. 48. Ginger Oleoresini 12.8 rngml“) 0.0305 x«. ;;: 0.0304 1 1 ‘ ‘l L : fl__»§_. “‘: ’~“‘f;1, 0.0303 5 0' :5 0.0302 0.0301 » 0.0300 2.03 4.03 6.00 8.120 10.00 12.00 14.00 16.00 13.00 20.00 22.00 24.00 Mnutes
  49. 49. New Stock Ginger Oleoresin 52.0 mgm| "‘ 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000 2.03 4.00 6.00 8.00 1000 12.00 14.00 10.00 10.00 2000 22.00 24.00 MnL. ‘.eS
  50. 50. Detection Limit of UV/ V is Detector Blank (Methanol) NVA St°°k Lir»: -ar Plat cl [N‘‘ALxi'Ab: c-11:1»: :2 E‘ JCC -C2 ‘ V M ‘ Q Gmuw nicer». --sin “I JJLLAL. ’ nmvplus E I! {HIE-C2 Gi : : ntcartnm § 2 : mE. (-,3 5!: - 52v~-g-ml .2 1 M552 :1 m. —.; .-. ;t; ag. .y 3 wt Cc ctccrusur saw :1’: -5‘: _ . V , , 0 w .1 3 _‘ y I 0.05571 - 0.0092 R‘ - 0.8597 '~""‘l'>'m= "" 0.34 ml-‘ NVA 0.170 mgcm*‘NVA um
  51. 51. Detection Limit of UVNis Detector Bhnk (Methanol) Stock ginger Oleoresin 12.8 mgml" Llnult l‘| at aO| ?lVA[ vu Ah: 5 uuE—u. ' V a DOE O2 1 one (12 9 norm: 1 not-a2 0,: )0E~oo ’lmuvL‘H 6.4 mgml" Ginger Oleore n 0 52.0 mgml“ Ginger Oleoresin
  52. 52. 0.85 Table of Data Obtained from HPLC Analyses . «‘miwto5 Arc-3 Cmc-antration .5 *3. Ahsorba were at maximum peak -4 3. (iingrr Olmrrxin 4483 16036 1 143957 2-11 140 51 I31 6762 3 DOE-04 5 00E—04 BDOE-03 6,00E-04 1 DOE-04 0 OCE*O0 u_. .8» 5,404 8 509 19 731 2 ‘-3 mi_ iris‘ l'I0 . im| iI . um1. 0.170 ? .‘3)1‘3 2—1(I| '/‘ED in L)'IZC~03 16372 4710-: ‘ISCE-03 19.1-109 6537 0,00C‘O0 7.847 6-170'] 2.DCE-03 13.234 531-$3 Q EDCE-0-1 16,125 13-150 ‘1 OC E-0-1 14122107 '. SE02 2'35EiF. O 1.U'I}C()Z 7-£672 3rUCE-03
  53. 53. Calculations of Response Factor The response factor for NVA is calculated as follows: K“, = (Cm x 100)I, ,., ,, mgI100mIIunit area c°"°°""'a"°"lcu N93 Absorbance at maximum W. ) (A, ,., ,) peak K, .,, ,(mgI100ml/ unit area ) 0.17mg ml-‘ 240730 a 6.00E-03 7.osE-os Stock NVA 0.85mgml" 1422107 38608 4.0054): 5.98E-05 Gragh of Km” vs Cum N 0 § 6_00E_04 Linear respdogse -= requires a i erence = 4.00504 - 53 ‘ Sfa"°s1 . of less than 2% for EC § 2_ooE-o4 — Linear (Senes1) any two points g 0.00E+00 -. E 0 0.5 1 y = 5E-05x + 0.0001 R2 = 0.0048 CNVA (mgml")
  54. 54. Response Factor for 6-Gingerol K[6]-G = KNVA x 298,38I293,41 = KNVA x 1,003 mgl100m| lunit area =6.00E-02 mgl100m| lunit area for 52.0mgImI oleoresin solution
  55. 55. Concentration of 6-Gingerol in 52 mg/ m101eoresin Solution - Calculated using: (AMG XKMG)/ C C= concentration of ginger oleoresin (1143957 area unitsx6.00E-02 mgl100m| lunit area )l52mglmlx100 =131.995 "/0
  56. 56. Error Analysis Equipment Tolerance Analytical Balance 0.00059 Sml Pipette 0.05mI 10ml pipette 0.04mi 10ml volumetric flask 0.04 ml 25ml Volumetric Flask 100ml Volumetric Flask
  57. 57. Error Analysis Continued Error in mass of ginger oleoresin " (0.0OO53+ 0.0005" =7.071x10‘5g = 7.071 x1 0"mg Error in concentration ofgingcr olcorcsin stock = ll. KinginI" 0012804389 mglml I‘-‘.07 I. lll'= m_u ‘Z'n‘Umg Hill. lbmli l0(lmI I‘ ' Error in concentration of of secondary stock solution =6.40mg/ ml x . (0.012804389 mg/ ml/12.8mg/ ml)? -0» (0.04m| /10ml)? = 0.026388 mglml . Error in percentage of oleoresin by mass =1.ae: i-/ .1 (io. oo0osgIss. szg)= x(o. oooosi1.o4)r) =1x10*°/ o = '0.026388
  58. 58. Sample Storage * Nonanoic acid Vanillylamide is thermally labile NVA was refrigerated when not in use. -' Ginger oleoresin samples were also kept refrigerated when not in use.
  59. 59. Requirements * Spike the sample and perform extraction in duplicate —Insufficient sample * Concentration of 6-gingerol * -could not be determined due to tailing and peak broadening in the only useable chromatogram
  60. 60. Methods used to Ensure Data Quality * Blanks * Used validated standard method * Extemal standard
  61. 61. #0- Problems Technical difficulties associated with HPLC Fluctuating pressure, solvent, baseline, curcumin, backflow of water into solvent Sample concentration needed to be increased Insufficient samples to make a spike
  62. 62. .°": l‘. °°! ’. -‘ Deviations from Validated Standard Method Concentration of NVA standard Mixing time Concentration of stock solution Initial Solvent Run time
  63. 63. Suggestions * Water trap should be attached to the degassing apparatus * ISO 9001 protocols regarding cleanup, definitive scheduling. r The mass of ginger rhizome required for analysis should be increased to compensate for the high detection limit of the detector.
  64. 64. Conclusions ° Of the results obtained, the values generated for the percentage mass of ginger oleoresin were of the highest quality(1.83°/ o1x1O'3°/ o) ° The validated method provided a systematic methodology of analysis which was easy to follow but the quality of the data generated is dependent on external variables. Some of which are beyond the analyst’s control.
  65. 65. Part II: Organic Components in Ginger by UVNis Spectroscopy
  66. 66. Extraction Method . Due to lack of time and availability of Samples the previously prepared ginger oleoresin was used
  67. 67. Trial 1:Vis/ Nir Spectrophotometer Pu8670 Vis NIR spectrophotometer - , -_, ., m L1,- ru'I" -. '': V . ’.‘'. I ’ '= I _III WW I_ F I . F / ‘ T T /
  68. 68. Instrumental Parameters Ir Wavelength -325nm * Response linear with respect to ginger oleoresin Ir NVA below the detection limit
  69. 69. Trial 2:UV/ Vis Spectrophotometer Spectronic 601 UVNis Spectrophotometer ,1 _ ‘ I it t W’ . _ . __-4/0 _i’£0,'_2", ‘ " I ‘ . ... _TI. ‘3 l I -—~—~. — . .._____ , ___________. . _S ' '7 u
  70. 70. Instrumental Parameters * Wavelength -282nm '* Cuvette- Quartz * Path length -lcm

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