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EFFICIENCY PARAMETERS AND RESOLUTION IN HPLC

Presented by

Jisna Sebastian I st year mpharmDept. of pharmaceutics.

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ContentsIntroductionTheoretical plate modelPlate numberHETPColumn efficiency ResolutionSeparation factorCapacity factorTotal retention timeNet retention timeDead volumePeak asymmetryCritical band pairTheories of chromatogrphy plate theory & rate theoryConclusionReference

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

It is defined as separation of mixtures of compounds due to differences in their distribution equilibrium between two phases, the stationary phase packed inside columns and the mobile phase , delivered through the columns by high pressure pumps

The Theoretical Plate Model of Chromatography

The plate model supposes that the chromatographic column

contains a large number of separate layers, called theoretical plates.

Separate equilibrations of the sample between the stationary and

mobile phase occur in these "plates".

The analyte moves down the column by transfer of equilibrated

mobile phase from one plate to the next. 4

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I t i s i mpor tan t to re me m ber t ha t the p l a t e s do no t rea l l y e x i s t ; the y are a f i gme nt o f th e i m ag i na t i on tha t he l ps us u nde r s t and the proc e s se s a t wo rk i n th e co l um n.

PLATE NUM BER

I t re f l e c t s i t s qua l i ty o f s e par a t i on and i t s ab i l i t y to produc e s har p , n ar row pe aks & ac h i e v i n g good re s o l u t i ons o f pe aks . 5

HETP[ Height Equivalent to a Theoretical P late ] A theoret ica l p late i s an imaginary unit of a co lumn, where distr ibut ion of so lute between, s tat ionary phase and mobi le phase, has at ta ined equi l ibrium. I t a lso cal led as funct ional uni t of the column.It can be of any height , which describes the ef f ic iency of separat ion . HETP = L / N i e , HETP = LENTH OF COLUMN / NUMBER OF THEORETICAL PLATESIf HETP is less , the column is more ef f ic ient .I f HETP is more , the column is l ess ef f ic ient .

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CO LUM N EFF ICI ENCY

Ef f i c i e n cy o f a c hrom atogr ap hi c p eak i s a me asu re o f th e d i s pe r s i on o f t he an a l y te band as i t t r ave l l e d through th e H P LC sys t e m and c o l u mn.

I t i s e xpre s se d by th e numb e r o f th e ore t i c a l p l a t e s .I t i s c a l cu l a te d by th e for mu l a ,

𝑁 = 16 ( / ) 𝑡𝑅 𝑤𝑏 2 = 5 .5 4 ( / 1 / 2 ) 𝑡𝑅 𝑤 2

tR i s th e re t e nt i on t i me m e as u re d f rom th e i ns tant o f i n j e c t i on , w i s the pe ak wi dth ,w i s de t e r mi ned by S D = σ i e , w= 4 σtR & w are measured in common units (cm or mm) , (min or sec

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If the retent ion t ime i s h igh and peak width i s narrow , i t g ives excel lent chromatogram. Eff ic iency can be increased by increasing the column length , reducing the column internal diameter .

Column efficiency 8

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RESO LUTIO N, Rs

The mo s t i mp or tant t h i ng i n H P LC i s t o obta i n th e opt i mu m re s o l u t i on i n the m i n i mum t i m eResolution is the ability to separate two signals. ie, separation of two constituents.

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ww

ttR RRs

Were tR1,tR2 & W1,W2 are the times &widths respectively of the immediately adjacent peaks.

RESOLUTION, Rs

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Th e Fu ndame n ta l Re s o l u t i on Equ at i on ( s hown) i nd i c a te s tha t re so l u t i on i s a f f e c t e d by thre e i m por ta nt p ar am e te r s : Se l e c t i v i t y ( Se par a t i o n fac t or ) Ef f i c i e n cy Re t e nt i o n (Capac i ty fa c t or )  In e quat i on , K i s t he re t e nt i on or c apac i ty f ac tor α i s t he s e l ec t i v i t y or s e par a t i on f ac torN i s th e Ef f i c i e nc yRs i s t he re so l u t i on

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RETENTION (CAPACITY) FACTOR (K)The retention (or capacity) factor (k) is a means of measuring the retention of an analyte on the chromatographic column.

A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase.

Determination of Retention Factor (k)

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SELECTIVITY (SEPARATION) FACTOR (α) The selectivity (or separation) factor (α) is the ability of the chromatographic system to ‘chemically’ distinguish between sample components. It is usually measured as a ratio of the retention (capacity) factors (k) of the two peaks in question and can be visualized as the distance between the apices of the two peaks.

High α values indicate good separating power and a good separation between the apex of each peak. 14

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T O TA L R E T E N T I O N T I M E ( t R 1 o r t R 2 ) : I t i s t h e t i m e r e q u i r e d b y a s a m p l e c o m p o n e n t t o m i g r a t e f r o m c o l u m n i n l e t ( s a m p l e i n j e c t i o n t i m e ) t o t h e c o l u m n e n d ( d e t e c t o r ) D E A D V O L U M E ( t 0 ) O R N O N - A D S O R B E D T I M E : - T i m e r e q u i r e d b y t h e i n e r t s u b s t a n c e t o m i g r a t e f r o m c o l u m n i n l e t t o c o l u m n e n d w i t h o u t b e i n g r e t a i n e d b y t h e s t a t i o n a r y p h a s e . N E T R E T E N T I O N T I M E ( t ’ r 1 o r t ’ r 2 ) : I t i s t h e d i f f e r e n c e b e t w e e n t o t a l r e t e n t i o n t i m e & t h e d e a d v o l u m e ( t 0 ) - t i m e t h e s a m p l e r e m a i n i n g w i t h t h e s t a t i o n a r y p h a s e .

t’R 1 =(tR 1 – t0) or t’R 2 =( tR 2 - t0) 15

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PEAK ASYMMETRY I n t h e i d e a l w o r l d a l l c h r o m a to g ra p h i c p e a k s wo u l d b e s y m m e t r i c a l ( o r G a u s s i a n ) . H o w e v e r, d u e to t h e e ffe c t s o f i n s t r u m e nt d e a d -v o l u m e , a d s o r p ti v e e ffe c t s o f t h e s ta ti o n a r y p h a s e a n d t h e q u a l i t y o f t h e c o l u m n p a c k i n g , p e a k s m ay o ft e n s h o w a ta i l i n g b e h av i o r. Ta i l i n g d e s c r i b e s a p e a k w h o s e ta i l p o r ti o n ( d i s t a n c e ‘ B ’ i n t h e d i a g ra m ) i s w i d e r t h a n t h e f r o nt p o r ti o n ( d i s t a n c e ‘A’ i n t h e d i a g ra m ) . A l s o , i f t h e s a m p l e c o n c e nt ra ti o n i s t o o h i g h o r i f t h e c o l u m n i s d a m a ge d a n d c o nt a i n s ‘c h a n n e l s ’ t h e n a f r o nti n g p e a k s h a p e m ay o c c u r.

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Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

CRITICAL BAND PAIRIt is a band pair in a chromatogram which has the smallest resolution value, while developing the method. Separation conditions are appropriately or systematically changed to improved upon this critical band pair. 17

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THEORIES OF CHROMATOGRAPHYTh e re are two t he or i e s to e xp l a i n c hrom at ogr aphy P l a te the or y - o l d er ; de ve l op ed by M ar t i n & Synge i n 1941 Rate t he ory - c u rrent l y i n us eProp ose d b y van De e mt er i n 1956Acc ou nt s fo r the dyn ami cs o f the s e par a t i onPLATE TH EO RYCol um n i s d i v i de d i n to a n umb e r (N) o f adj ace n t i mag i nar y se gme n ts c a l l e d th e ore t i c a l p l a t e s .Wi t h i n e ac h t he ore t i c a l p l a t e ana l y te ( s ) c ompl e te l y e qu i l i br a te be twe e n s ta t i ona ry phas e and mobi l e phas e

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TH EO RI ES O F CH RO M ATO G RAPH Y The re are two th eor i e s t o e xp l a i n c hromat ogr aphy P l a te the or y - o l de r ; de ve l op ed by M ar t i n & Synge i n 1941 Rate t heo ry - cu rre n t l y i n us eProp ose d b y van De e mte r i n 1956Acc oun t s fo r t he dyn ami cs o f the s e par a t i onPLATE TH EO RYCol um n i s d i v i de d i n to a n umb er (N) o f adj ac e n t i mag i nar y s e gme n ts c a l l e d th eore t i c a l p l a t e s .Wi t h i n e ac h t he ore t i c a l p l a t e ana l y te ( s ) c ompl e t e l y eq u i l i br a te b e twe e n s ta t i onar y phas e and mobi l e p has e

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PREDICTIONS OF PLATE THEORYBand spreading - the width of bands increases as their retention time (tR) or retention volume (VR) increasesThe smaller HETP, the narrower the eluted peak RATE THEORYIn the rate theory, a number of different peak dispersion processes were proposed and expressions were developed that described • the contribution of each of the processes to the total variance of the eluted peak • the final equation that gave an expression for the variance per unit length of the column The processes proposed were •Eddy diffusion •Longitudinal diffusion •Resistance to mass transfer in the mobile phase •Resistance to mass transfer in the stationary phase

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Th i s Th e ory G i ve s

•more re a l i s t i c de s c r i p t i on o f the pro ce s s e s th a t wor k i ns i de a c o l u mn

• Take s ac c ount o f th e t i me tak en for t he s o l u t e to a t ta i n e qu i l i br i u m be twe e n t he s t a t i onar y and m obi l e phas e

• Th e re su l t i ng b and s ha pe o f a c hromat ogr aphi c pe ak i s the re fo re a f f e c t ed by th e r a t e o f e l u t i on

• I t i s a l s o a f f e c t e d b y the d i f f e re nt pa th s ava i l ab l e to s o l u te mo l ec u l e s a s th e y t r ave l be twe e n par t i c l e s o f th e s t a t i onar y phas e 21

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THE RATE THEORY OF CHROMATOGRAPHYThe rate theory has resulted in a number of different equations At normal operating velocities it has been demonstrated that the Van Deemter equation gives the best fit to experimental data The Van Deemter EquationH = A + B/u + u [CM + CS]

A: random movement through stationary phase (Eddy diffusion)B: diffusion in mobile phase (Longitudinal Diffusion)C: Resistance to mass transfer in mobile & stationary phaseH: plate heightu: average linear velocity u = L/ tM

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T E R M A : E D D Y D I F F U S I O NA p r o c e s s t h a t l e a d s t o p e a k ( b a n d ) b r o a d e n i n g d u e t o t h e p r e s e n c e o f m u l t i p l e f l o w p a t h s t h r o u g h a p a c k e d c o l u m n .A s s o l u t e m o l e c u l e s t r a v e l l e d t h r o u g h t h e c o l u m n , s o m e a r r i v e d a t t h e e n d s o o n e r , t h e n o t h e r s s i m p l y d u e t o t h e d i f f e r e n t p a t h t r a v e l l e d a r o u n d t h e s u p p o r t p a r t i c l e s i n t h e c o l u m n t h a t r e s u l t s i n d i f f e r e n t t r a v e l d i s t a n c e s . So widening of the peak width occurs.

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TERM B: LO NG ITUDI NAL DIF FUSSI O NA ban d o f ana l y t e m o l e c u l e c onta i ne d i n the i n j ec t i on so l v en t w i l l t e nd to d i s per s e i n e ve r y d i re c t i on d ue to t he c onc e ntr a t i on gr ad i e nt a t the oute r e d ge s o f the b an d .

TERM C: RESIS TANCE TO M ASS TRANSF ER

Anal y t e t ake s a c e r ta i n amou nt o f t i m e to e qu i l i br a te b e twe en the s ta t i onar y and mo bi l e phas e . I f th e ve l oc i ty o f th e mobi l e phas e i s h i gh , and th e ana l y te has a s t rong a f f i n i ty f or t he s t a t i onar y p has e , t he n the ana l y te i n t he m obi l e phas e w i l l m ove ahe ad o f the an a l y te i n the s t a t i onar y phas e . Th e b and o f ana l y te i s broade ne d . The h i gh er the ve l oc i t y o f mo bi l e phas e , th e wor s e the b ro ade n i n g be c om e s .

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Mobile phase mass transfer term (CM/u)

Stationary phase mass transfer term (CS/u)

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From the ab ove f i gu re , we c an unde r s tan d th a t ,

Ed dy d i f f us i on , the A t e r m, i s c aus ed by a tu rb u l e nc e i n th e so l u te f l ow pa t h and i s m a i n l y unaf f e c t ed by f l ow ra t e .

Lon g i tu d i na l d i f fus i on , th e B t e r m, i s th e move m en t o f an ana l y te m o l e c u l e o utwar d f rom th e c e nte r to the e d ge s o f i t s ban d . H i ghe r c o l u mn ve l oc i t i e s w i l l l i m i t t h i s out war d d i s t r i bu t i on , ke e p i ng the band t i gh te r .

M ass t r an s f e r, th e C t e rm , i s th e m ove me nt o f ana l y t e , or t r ans fe r o f i t s m as s , be t we e n the mob i l e and s ta t i onar y phas e s . Inc re as e d f l ow has be e n ob se r ve d to w i de n ana l y te b ands , or l ower pe ak e f f i c i e nc i e s .

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CONCLUSION

Di f f e ren t p ar ame te r s wh i c h i s re l a t e d to the ope r a t i on o f th e who l e ch romat ogr aphi c s y s t e m c an be m oni t ore d and i n ve s t i ga te d t o f i n d out the i n t egr i ty and re l i ab i l i t y o f t he who l e H P LC sys t e m s .

Th es e par am et e r s are us e d to de te r m i ne c har ac te r i s t i c c h romat ogr aphi c par am et e r s , suc h as th e n umbe r o f e f f e c t i v e th e ore t i c a l p l a t e s , re s o l u t i on , a s ymm e tr y, de te c t i o n l i mi t and s e l e c t i v i t y.

Af t e r t he c he ck i ng o f the p ar am e te r s , the s y s t e m i s the n on l y c an be de c l are d s u i t ab l e i f the re spon se s are w i t h i n pe r m i t t e d l i m i t s

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REF ERENCES

1 .H i gh pe r for m anc e l i q u i d ch romat ogr aphy by P.D SETH I .

2 . Fu ndam e nta l s o f a na l y t i c a l c h em i s t r y, by Skoog ,We s t ho l l e r , Crouc h pg 982 .

3 . I ns t r um e nta l m et hods o f an a l ys i s by Wi l l ard , M e r i t t de an , Se t t l e s e ve nth e d i t i on .

4 . In s t r ume nt a l an a l ys i s by G URDEEP R CH ATWAL an d SH YAM K ANAND.

5 .G oog l e s e arc h29

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