Beyond HPLC…. Additional techniques for the analysis of your starch-derived products and transformation processes

07 June 2011, Rafael F. Sala , Ph.D., Scientist II Applications Research

Genencor (A Danisco Division), Palo Alto, USA

Outline

• Introduction

• What other techniques besides HPLC?

• H NMR

• MALDI- MS

• Capillary Electrophoresis

• Follow up of saccharification

• End of Fermentation analysis

• High Dextrose syrup analysis

• Conclusion

• Questions

How to monitor starch processing enzymes?.

Production of Glucose from starch: Alpha-Amylase and Glucosidase

Glucoamylase: degrades starch by breaking the 1,4 and, to a lesser extend, 1,6 bonds from the non reducing ends of poly/oligosaccharides (i.e. exo-glucosidase)

IntroductionUse of HPLC is ubiquitous in the industry for analysis of starch derived products and follow up of processes. (SEC, IEX, ion exclusion, RP, NP, HIC, HILIC, etc)

Other analytical techniques are used, some of them very often, some rarely

• Calorimetry, Specific Gravity, Viscosity, %humidity, DE, Iodine, Brix, etc

• Gas Chromatography (GC), GC-Mass Spectroscopy (GC-MS)

• Near Infra Red Spectroscopy (NIR)

• Nuclear Magnetic Resonance (NMR)

• Matrix Assisted Laser Desorption Ionization MS (MALDI-MS)

• Capillary Electrophoresis (CE)

• Ultrasonic Spectroscopy

• Quartz Crystal Microbalance (QCM)

• Other

The ‘eyes of the chemist’ :1H NMR spectroscopy…. too much physics for a chemist

• Based on behavior of nuclei with non-zero magnetic moment (1H, 13C, 15N, 31P) on a high magnetic field

• Information rich technique, signals characterized by their frequency (chemical shift), intensity, fine structure and magnetic relaxation properties.

• Most used 1H NMR, allow the mapping of each proton in a molecule based on its different magnetic micro-environment.

• Used in ‘metabolomics’ and also amenable for ‘in situ’ monitoring of transformations. Non destructive.

1H NMR analysis: initial soluble liquefact…

Non-reducing sugars

-H1-6,nr

-H1,r-H1,r

Reducing sugarsAnomeric Region

O

O

OHO

OH

OH

HO

O

OH

OH

OH

H

-H1-4,nr

OOHOHO

OH

OH

H

O

OH

OHO

OH

OH

H

H1-r H1-r

• 1H NMR allows the direct observation of the components of the saccharification mixture. The

anomeric region is particularly useful to evaluate the presence of fermentable sugars and reduced

sugars.

• Can follow up progress (kinetics) of saccharification ‘in situ’, but can not follow up fermentations

(CO2 evolution)

-H1-4,nr

Use of 1H-NMR in the analysis of starch processing

Expected 1H NMR signals on Liquefact• Reducing ends:

• Anomeric : H1 (5.2 ppm) and H1(4.6 ppm)

• H2 (3.26 ppm) ; H4 (3.41 ppm)?

• Other protons: undefined (3.5 to 4.0) ppm

• Non-reducing ends:• Anomeric : H1-4 (5.40 ppm), no H1-4

• H1-4 in branching (hump 5.3-5.38 ppm)

• H1-6 (4.96 ppm)O

O

OHO

OH

OH

HO

O

OH

OH

OH

H

O

O

OHO

OH

OH

HO

O

OH

O

OH

O

OH

OH

OH

HO

H

OOHOHO

OH

OH

H

O

OH

OHO

OH

OH

H

H1-r

H1-4-nr

H1-6-nr

H1-r

Reducing anomeric protons

Non-reducing anomeric protons

1H NMR analysis Soluble Liquefact

‘Organic Acid’ HPLC analysis

End of Saccharification…almost all glucose

OOHOHOH

OH

OH

H

O

OH

OHOH

OH

OH

H

H1H1

15 min

Start

50 min

205 min

1H NMR follow up of GA effect on soluble part of Liquefact

1H NMR follow up Pullulanase on soluble part of Liquefact

2 min

10 min

1 h

24.5 h

H1-6nr

H1-4nrClose to 1-6

H1-4nr

H1rH1r

How sensitive the 1H NMR is? …

Standard High resolution NMR can be sensitive enough to detect micrograms levels (in the NMR tube)

A rough approximation of the detection limit of 1H NMR is 1-10 mg/ml for small molecules (e.g. for glucose aprox 5 mM or 0.1%).1H NMR of an incomplete saccharification with 0.5% internal standard DSS

….looks like pure glucose, however upon a closer look (expansion)….

DSS @ 0.5%

…same spectrum zoomed (20x)

I can ‘see’ that the saccharification is incomplete….

H1,4 nr unreacted

?

DSS

Saccharification reaction follow up with 1H NMR

• An array experiment with quantification of each signal (integral value) allows the monitoring of the changes on liquefact after addition of glucoamylase or any other enzyme

• Array is made by changing the pre-acquisition delay to desired time intervals

• Monitor only the anomeric region gives plenty information

Saccharification reaction follow up with 1H NMR

A detailed progress curve can be drawn using 1H NMR.

Ratio of non-reducing / reducing sugars or extent of hydrolysis (reducing / total) can be monitored.

Reducing sugars can be correlated to DE numbers.

More information can be elicited as the evolution of particular protons that make up the reducing and non-reducing components can also be monitored.

Amount of enzyme can be tuned up for transformation on a desired length of time

…a more detailed view: monitoring the anomers…

• 1H NMR allow observation of the hydrolytic cleavage for each anomer (and )

• The results show that the anomer is formed preferentially during the process and then equilibrate (mutarotate) to the standard mixture (36/64).

• This is consistent with an ‘inverting mechanism’ known for glucoamylases.

…..and the branching number

Changes on hydrolysis of H1,4 and H1,6 can be followed up

Change on the degree of branching (defined as the ratio H1 nr total / H1,6 nr) can also be monitored.

Comparison among enzymes or blends on the effect on branching points can be studied using this technique.

1H NMR studies on model substrate: Panose

26h follow up, only 3 anomeric protons H 1,4nr, H1,6nr, H1r ( and

OHO

O

OH

OH

OH

H

OOH

OH

OH OH

HOH O

OH

OH

OH OH

H

Maltose Glucose

OHO

O

OH

O

OH

O

OH

OH

OH

HOH

H

OOH

OH

OH OH

H

PanoseAp

Bp

Cp

Am

Bm

k1k2

Progress curve analysis …Maltose by difference

Simulation suggest k2=2.5 k1

-0.20

0.30

0.80

1.30

1.80

2.30

2.80

0 200 400 600 800 1000 1200 1400 1600 1800

Panose hyd. WT

Glucose gen. WT

Maltose rem. WT

Time(min)

EOF analysis

By the time EOF is collected (55h) there is negligible production of Ethanol from fermentable sugars.

However the DP4+ peak is still a major peak on the HPLC trace

EOF was collected, filtered, extracted with organic solvent and lyophilized to facilitate its analysis

Liophylized soluble part of EOF was analyzed by HPLC & NMR

What is in the DP4+ peak?…but also, what is on the DP2 / DP1 regions?

Lyophilized EOF residue

DP2s

DP1sNo Ethanol

HPLC on HPX-87H column of the EOF residue

1H NMR of liophylized EOF helps to identify metabolites...

Single anomeric signal ?

1H NMR show unambiguously that all fermentable sugars have been depleted from the soluble part of liquefact.

As observed on the HPLC Glycerol and Lactic acid are shown in the 1H NMR analysis

A signal in the -anomeric region of the 1H NMR suggest the presence of a non-fermentable sugar of unknown structure.

Glycerol

fermentable sugars

Lactic acid4 . 05 . 0

Clear

?

Lactic acid

Start liquefact

?? ?

Identifying Trehalose as the unknown sugar at EOF

Trehalose

EOF

-1,1 Gluc

The single anomeric nature of the sugar (no -anomer) hints at Trehalose as the ‘undigestable’ disaccharide.

1H NMR confirms Trehalose as the unknown sugar residue at the EOF

OHO

O

OH

OH

OH

OHO

OH

OH

OH

Identifying other metabolites….

Lactate Propylene glycol,2,3 butanediol?

Trehalose

Trehalose

Trehalose

Phytic acid or Phosphate Inositols?

?

Lactate

Glycerol

Analysis of High fermentable sugar syrup (High Glucose syrup)

High Glucose syrup (96+%) needed for Sweeteners applications

Problem with HGS is formation of reversion products at high Glucose concentration: Isomaltose and Maltose.

Structurally similar saccharides are difficult to separate and detect:

• They are polar but usually not charged (unless high pH or has charged groups).

• Detection usually by Refractive index but poor sensitivity and not amenable to gradients.

HPLC separation of Maltose/Isomaltose not trivial, need a technique which could deliver a better resolution

GC is an option but is there other options?.

Isomaltose Maltose

HPLC (Organic Acid column) of conventional process

Putting your HPLC on steroids: Capillary electrophoresis

• CE = Gel Electrophoresis + HPLC

• CE employs narrow-bore (20-200 mm i.d.) capillaries to perform high efficiency separations of both large and small molecules.

• Separations are facilitated by the use of high voltages, which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary.

• Analytes can be separated by mass, charge or isoelectric point

• Usually ‘state of the art’ detection technology is used for registering the electropherogram

• Uses small amounts of reagents and sample.

• Resolution as good or better than capillary GC.

©Figures: www.chnola‐research.org and Harris, D.C., Quantitative Chemical Analysis, 7th ed.,W.H. Freeman Co., NY, 2007.

FACE: Labeling reducing sugars for separation and detection

Detection with Laser-induce fluorescence allows high resolution

Figure:  O’Shea M.G., et al. Carbohyd. Res. (1998) 307, 1‐12 

200

250

300

Standards

MaltoseIsomaltose

Maltotriose

Isomaltotriose

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0

50

100

Conventional process

% ratio: unknown: 2

isomaltose: 52

maltose: 46

IsomaltoseMaltose

Maltotriose

CE analysis on high glucose syrup for conventional process

MaltotrioseMaltoseIsomaltose

-Glucose + other-reducing ends

-Glucose + other-reducing ends

1H NMR confirms FACE analysis (anomeric region only)

Use of 1H NMR in Biomass analysis….

• Biomass treatments produces complex mixtures

• 1H NMR anomeric analysis can be used on analysis of hydrolysates.

• Ratios of different sugars on mixture is important for SSF processes.

DSS

H1-non-reduced region

H2-reduced (Glucose)

H1-Glucose

H1-reduced (Glucose)

H1-mannose

-xylose

-xylose +-galactose

H1-mannose

H1-galactose

H1-Glucoronic acid

-L-arabinose

H1,6-?

1H NMR of pretreated Biomass

Another tool…. MALDI-MS

• Matrix Assisted Laser Desorption Ionization (MALDI) was one of the first MS techniques available for the analysis of proteins due to its ‘gentle’ ionization.

• It is also useful for the analysis of polydisperse oligosacharides such as the ones found in starch processing

• An alternative to other high resolution chromatographic techniques such as FACE and HPAEC-PAD.

Time-of-flight (TOF)Mass Analyzer

Follow up of SSF process: Start, 6h, 18h…

6h

18h

Start Lqfct

…25,43, 55h (EOF)

55h

25h

43h

55h

Conclusions

HPLC is the workhorse for analysis of saccharides in saccharification / fermentation processes but has limitations on the information it provides.

Other analytical techniques such as NMR, CE and MALDI-MS can provide additional information to monitor a process or to detect, isolate and quantify a component.

1H NMR is particularly useful to monitor the branching point evolution during a saccharification reaction.

MALDI and CE are useful to analysis of monomer distribution of polysaccharides.

Other techniques are being developed that can assist in a better understanding of processes associated to the Grain / Biomass industry.

1H NMR sensitivity – sample requirement